Staining - Science method

I used TEM to observe damaged bacteria due the use of antibiotics. However, I am not sure whether the image I took is contamination due to staining or the damaged bacteria (E. coli).

Thank you for any help!

I'm doing an IF experiment to show the GBP (guanylate binding protein) protein level (cytosolic protein).

However I'm having trouble doing my experiment, the fluorescence signals are expressed in the background, not at the protein location.

Other papers staining the GBP protein show that only specific areas with GBP are stained like dots.

The protocol below was used to stain the GBP protein in the raw cell line.

1. Fixation: Incubate the sample with 4% PFA for 15min at RT. Wash in PBS 3 times.

2. Permeabilization: Incubate the samples for 10min with 0.1% PBST(0.1% Triton X-100 in PBS). Wash in PBS 3 times.

3. Blocking: Incubate the cell with 5% goat serum with 0.1% PBST for 40min and wash 3 times in 1% goat serum (PBS).

4. Staining: Dilute 1’ Ab in 1% goat serum in PBS and stain at 4’ for O/N and wash in 1% goat serum with PBS 3 times. Dilute the 2’ Ab in 1% goat serum in PBS and incubate at RT for 1h in the dark. Wash in 1% goat serum with PBS.

5. Nuclear counterstaining and mounting: Incubate the antifade DAPI solution for 2-4min at RT.

I am using these antibodies,

1. GBP1-5 (Santa Cruz, sc-166960): dilution 1:200

2. Goat anti-mouse IgG (H+L) Highly Cross-adsorbed secondary antibody Alexa Fluor 488 (Invitrogen): dilution 1:400

Which steps do you think are creating the wrong staining image?

I attached the image that I made.

Thank you.

I shake microglia (200rpm, 2h) from P0-P3 mice after 12-14d culture of primary mixed glia. After another 3-4 days culture, I stain the cells with Iba1/GFAP/Oligo2 antibody, but I find that all these markers can stain every cell. Has anyone encountered this? What is the possible reason?

My IF protocol: 1. wash three times with PBS, 2. fix cells with 4%PFA and 120nM surcose in PBS for 15min at RT, 3. 3 x 5min wash in PBS, 4. block cells with 3% donkey serum, 5. incubate cells with 1 antibody over night at 4℃, 6. 3 x 5min wash in PBS, 7. incubate cells in 2 antibody for 1h at RT, 8. 3 x 5min wash in PBS.

As we are finding difficult to secure the Trypan Blue supplies in Colombia, i would like to know if there is any alternative reagent to Stain the PBMC cells.

Hello,

I am wondering if anyone here who performs SDS-PAGE has seen this before on their gels post-staining? We make our 12% tricine gels in house, fix them in 25% isopropanol 10% acetic acid, then stained overnight in Coomassie G250 35mM HCl. The gels are then destained in distilled water. We are noticing what seems like "halos" or zones of white around our proteins in the gels. I have images and notes attached regarding the issue. When the peptide is in its neat form, it is in a 1M imidazole, 500mM NaCl, 20mM Tris buffer at pH 8. The peptide has a final concentration of 200mM imidazole when it is in its 1/5 diluted form. We have seen this effect many times before, but are not sure what is it causing it. Is it perhaps due to the presence of imidazole; can the imidazole, or maybe just an overall high salt concentration, cause this effect? We use fresh running buffer, fresh fixative and fresh gel reagents (e.g. new aliquots of APS) for each run. Coomassie is reused and made fresh every month and a half; the Coomassie used here is less than a month old.

Any input or words of wisdom would be greatly appreciated! Many thanks in advance.

Leisha

Good day, everybody. I have stained sagranin O rats bones with an intra-articular fracture. In all figures in papers it should be stained blue (bones) and red (cartillage). But as it seen at the figures bone structures stained pink-violet.

Protocol is so:

1. Deparafinize and rehydrate.

2. Weigert's Iron Hematoxylin stain for 10 min.

3. Wash in tap water for 10 minutes.

4. Fast green stain for 10 minutes.

5. Wash fast in 1% acetic acid for 10 seconds.

6. Stain Safranin O for 5 minutes.

7. Dehydrate and clear in xylene.

May be problem in protocol? May be somebody can recommend another protocol?

I need stain lipid droplets in an immunofluorescence for macrophages. I read that I can use Sudan III to do this, but I don´t find any procedure. The only one is this , but the author don’t describe the procedure of the stain.

I am doing multiplex immunofluorescence assay on FFPE section. I used CD3 to identify T cells and observed that some of T cells stained with CD3 showed nuclear staining instead of membranous. I am not sure why the staining pattern is nuclear for CD3? is it possible to see nuclear staining with CD3?

I just established a cell line after long-term term culturing and would like to screen for contamination. I only have Hoechst 33342, and I would like to know whether it can be used for mycoplasma screening.

I need to perform staining of A549 lung cancer cells using Alexa flour 594 phalloidin. Can anyone explain the protocol

Hi all,

I’m staining murine brains for degraded myelin basic protein (sometimes called cryptic MBP, also known as MBP69-86) and would like input on what we are seeing. Basically, it doesn’t look like there’s a positive signal where undegraded MBP would be, but is intracellular in the cell somas, and seems to be in neurons, which shouldn’t phagocytose it. Had anyone worked with this in the CNS before, and has an idea on the mechanisms behind what is happening/how we could quantify moving forward? Some early sample images are attached, and we are using cryopreserved brains and antibody cat# AB5864 from Sigma.

Thank you!

I want to stain some of my mouse brain sections with Cresyl Violet.

The mouse brains (not perfused, no PFA/Formalin used) were fresh frozen in OCT and cut in the cryostat at 15μm thickness. The sections were collected on normal slides and stored at -20℃.

Could you please help me with the staining protocol?

Does anyone have a live/dead stain that works for HFF cell lines?

I'm trying to perform immunofluorescence staining of Dynein and kinesin on human skin, but I don't know how it should appear, and I looked through published apers and I didn't find any. Any help? any body did this staining on human skin?

I did a Hoechst stains to check whether my cells were contaminated with Mycoplasm. Please advise or assist.

I'm trying to find a good protocol to co-stain Alzheimer's disease tissue with thioflavin S and 4G8 antibody with IHQ. I haven't found any protocol to perform these two stainings together. I do not want IF, I need to use HRP-secondary antibody. I'd really appreciate it if anyone has suggestions.

Thanks!

How long does it take to dye?

How to determine the concentration of dye?

If it is detected by ELISA, will the staining method be different?

We did in vitro differentiation of MSC cells into osteogenic and adipogenic lineage cells using STEMCELL kit. The protocol recommended to use culture-expanded human MSCs between passages 1 - 4 but cells of passage number 8 were induced to form osteoblast and adipocytes by culturing in osteogenic and adipogenic induction medium for 14 days, 24- well plate was seeded by 10000cell/cm2 and incubated for 48h to reach 80% confluent, then the media was replaced with complete MesenCult™ Osteogenic and adipogenic Differentiation and medium was changed every 2-3 days Osteogenic and adipogenic differentiation was visualized by staining with Alizarin Red S and Oil Red O staining, respectively. It seems that lipid accumulation happened for negative control (no differentiation media) and also the same pattern for osteogenic negative control.

I was wondering if someone had thought of a better and more time efficient solution.

Thanks

Hi all,

I'm trying to evaluate the amount of viable E. coli cells in a colture.

I'm going to use the Nuclear Green LCS1 and I will use a plate reader to evaluate the fluorescence emitted by the cells.

Is Nuclear Green LCS1 permeable to prokariotic cells?

Thanks for your help

hematoxyline and eosin are routin stain

I have done DAPI staining in untreated and drug-treated EA carcinoma cells using 96-well plate. The procedure included only pbs washes, fixation with paraformaldehyde and incubation of each wells with DAPI (50ul of 1:1000 dilution). Haven't done any permeabilization process, but, still got stained images. Is it proper to do so?

Hi everyone!

We isolated and sequenced our nuclei, and performed hashing prior to sequencing by combining four samples in a single batch, with each sample being stained with a different TotalSeq B hashtag antibody. I am curious whether the 10X Cloud Analysis tool can support the demultiplex Cellranger pipeline for our specific case.

Thank you for your help.

I am looking to stain blood cells in suspension in order to use in a flow cytometer. Is there protocol for staining suspended cells in culture media or PBS? Thank you!

there are many types of carbohydrates(glycoprotein ) :neutral and acidic glycoprotein

Hi,

I would like to know how people usually handle phalloidin dye (to stain actin) during immunochemistry? I know that is comes from the deadly mushroom Amanita phalloides. I have read the security data sheet that says that it is fatal if swollen, if in skin contact and if inhalated (https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2FSDS%2FT7471_MTR-NALT_EN.pdf).

However, I have read in the user guide of this compound that the amount of toxins in each vial was lethal "only" for a mosquito. I am then a bit puzzled and I am not sure how to handle this product.

Do you handle it under a fume hood using gloves and eye protection? Or is there something more to do to be safer?

Thanks for your reply.

Planning to do DAPI staining using fluorescence microscopy. Thought of seeding the cells in 6-well plate. What all things should be considered while doing the assay? Does anyone have protocol for the aforementioned?

My protein of interest is fused with MBP tag, and it does not contain tryptophan but MBP does. After factor Xa digestion, my protein was separated from MBP, and I eluted my protein while MBP is still in the MBP column.I ran SDS-PAGE with TGX stain-free gel.

Theoretically, my protein should not be visualized in Bio-Rad ChemiDoc imaging system machine because it doesn't have tryptophan. Stain-Free gels (Bio-Rad) contain trihalo compound and they interact with tryptophan residues of your protein and show fluorescent signal by UV detection.

There is no MBP contamination as it stays in the MBP column and it will only be eluted by maltose. What could be the reason?

Thank you in advance!

Hello,

i already have in the lab this live cell stain suitable for flow cytometry (CytoTrack Green 511/525 BIO-RAD) and i was wondering if i could use it to stain live spheroids which will then be analyzed and imaged by confocal microscopy. Has anybody already used this product for applications besides flow cytometry? Thanks in advance for the help

Tried with 7.5%, 10%, 12% and 15% gel percentages.. Protein concentration was checked through biuret and it is about 1.7mg/ml.. Used coomassie blue for staining (1hr) followed by destaining for overnight.. Any suggestion will be very helpful..

I would like to do a FACS analysis with BrdU-7aad staining, how long should I incubate with BrdU if my cells doubling time is 48hours?

Hi everyone,

We want to start performing some FluoroJade C stainings at the lab but the product that other labs have recommended is longer available (AG325 from Sigma-Aldrich). Do you have any other recommendation? We are in Spain, so brands from Europe or American ones with European distributors are preferred.

Many many thanks!

I have been running gel zymography (7.5% polyacrylamide co-polymerized with 1% gelatin) loaded with 250 µg of protein / well from colonic lysate with the hopes of visualizing MMP-2 and -9. I had a few good gels, but recently I have been getting these white spots on my gels (see attached) post-Coomassie staining and de-staining, following a protocol by Frankowski et al., 2012 (doi: 10.1007/978-1-61779-452-0_15).

Can someone explain where these big spots are coming from? It makes it impossible to quantify my images. I thought it was the Coomassie itself, perhaps due to chunks of undissolved Coomassie; however, I filtered the stain and still have the same problems. My most recent gel looks the exact same as this image, with the same two white circles.

Additionally, is using 1% gelatin appropriate? I have seen researchers use 0.1-0.2% gelatin co-polymerized with the polyacrylamide, but the source I listed above used 1%.

Thank you!

May some of you performed staining for immunofluorescence of PBMCs in 96-well plate? As the cells are not going to be cultured, only stained in these plates for confocal microscopy, we are planning to centrifuge (600 g, RT, 6 min) cells in 96 well plate to attach them to the surface of the plate, however I am not sure if this is enough to attach them. I know there are coating plates, but for decreasing costs we are searching alternatives.

We are isolating hepatocytes from mouse liver and trying to culture them in 6 or 24 well plates. The isolation is carried out according to the protocol and plating is done using William E medium with 10% FBS and 1% PEST. We use to coat the plates with collagen 1mg/ml. But after 3 hours and more the cells are loosely attached to the plates. We are sure these cells are hepatocytes because we stained them with the appropriate markers. Maybe something is missing in the medium which is essential for attachment?

Hello,

I am measuring multiple different parameters in cells using fluorescent channels in flow. I would also like to assess well viability. Given that I take the same volume from each well, and do not define a stopping event, can I compare live events between wells as a viability measure? I understand that not all cells that appear live in flow are viable, thus the use of viability stains. Do such stains have to be used to generate viability data from flow?

Thanks!

Hello Everyone,

I had ordered Recombinant Alexa Fluor® 488 Anti-GFAP antibody from abcam to stain specifically astrocytes. How ever, this antibody is staining other cells too. Does anyone have a similar experience with this GFAP antibody with regards to non specific staining?

We seeded 2,000 Dermal Papilla cells in 96 well-plate and treated the cells

for 24, 48, and 72 hrs in order to check the proliferative effects of our compounds.

Then we stained them with 0.05% crystal violet at the different time points and we found a purple plaque as shown in the image.

I would like to ask that

1. What is the plaque??

2. Where does the plaque come from?

I am aware that immunohistochemistry is a qualitative or semi-quantitative technique, but I performed several anti-CD163 immunohistochemistries in order to study the presence/absence of M2 macrophages in patients with several conditions. However, I have never quantified the result of immunohistochemistry before, so in the optical microscopy images that I took I see several brown marks (I used DAB for detection) but I am unable to determine if those marks are unspecific or if they are actually staining macrophages. Is there a way of correctly determining which of those DAB stains actually determine macrophages?

Thank you in advance.

Dear all,

I was puzzled by my present TEM results: PTA staining showed a significant low frequency of AAV empty capsids compared with that of UA staining. I used the same sample and conditions (only different in staining reagents), and I really dont know why UA staining could show so many VP with black dot within (empty), however PTA just got many bright full capsids.

And I rarely noticed AAV empty capsid was measured using PTA as staining solution.

Does anyone know the possible reasons?

MANY THANKS!

Dear colleagues!

I’m working with mouse splenocytes now to elaborate Treg suppression assay. But I have a trouble at the CFSE staining step. After my initial staining of total splenocytes or some sorted populations of cells from them (naïve CD4 T-cells), next day I observe the initial peaks of CFSE shift to the left with the loss if CFSE intensity ten times. So, it is not a first division when the CSFE intensity should drop 2 times only. It happens in a sample stimulated with CD3/CD28 beads and in a non-stimulated sample. Further 3 days afterwards I observe distinct division CFSE peaks in a stimulated sample. But I do not like that initial CFSE shift.

First, I thought that it was due to unsynchronized cell cycle of splenocytes and tried to synchronize them before CFSE staining keeping the cells not in a full media (RPMI + 10%FBS + 50uM ME + 25mM HEPES + 1mM Sodium Piruvate + 1% Glutamax + 1%Pen-Str) but in a media deprived of IL-2 or in a media with low 1%FBS or in a media with 1%FBS and without IL-2. Nothing changed. Next day my initial CFSE peaks again 10 times shifted.

Where is a mistake? Or is it a standard phenomenon? I add a file with the demonstration of that shift.

My protocol of CFSE staining is as follows: I wash cells with (DPBS+0,1% FBS) two times. I know that someone recommended to get rid of any traces of FBS at this step but others recommend to add FBS to splenocytes as they are very sensitive cells and quickly die without any FBS. Then I stain them for 5 minutes at 37C again in (DPBS+0,1% FBS) and then wash with cold RPMI with 10%FBS – two times.

I appreciate it greatly any tip and possible explanation!

I previously tried isolating rhizobial strains in YEMA congo red media, choosing the isolate which dint take the congo red stain. But after molecular characterization with sanger sequencing (16S rRNA partial), it was found that most of the isolates dint belong to any of the rhizobial genera. Can anyone suggest me any rapid method for isolating a higher number of rhizobial isolates?

I performed fluorescence stainings on brain formalin-fixed paraffin embedded samples (zebrafish). I stained TH+ neurons and counterstained with DAPI.

In tissues coming from 2 specific animals, DAPI (diluted 1:1000) is not working. For all the other samples, belonging to other animals, DAPI is working perfectly.

I am positive that this outcome is not user-dependent, because in the same session I stained many slides, and in some of them the DAPI worked, in some others it didn't. TH staining worked perfectly for all tissues.

P.S. I performed an antigen retrieval with citrate buffer pH 6.0 for 15 minutes at 95°C in a water bath.

Please help! I don't know what could have happened!

I'm trying to measure the ROS production from RAW 264.7 cells by using DCFH staining and analyst by flow cytrometry. The problem is the level of ROS production in control cells was high. I'm not sure what am I doing wrong. Can anyone help me solve this problem?

I use MCF7 cells to overexpress my protein of interest tagged with RFP using Polyethylimine. Then I stain my cells with monodansyl cadeverine(Specific for Autophagolysosomes)(50mM) which is dissolved in DMSO, following this I fix my cells with 4% paraformaldehyde, then followed by Hoechst staining. I'm not able to pick up RFP fluorescence right after my MDC staining. RFP fluorescence could not be captured either in the control which is over expressing RFP (added 200ul of DMSO per well without stain) or in the treated which is over expressing RFP tagged protein, right after the addition of staining solution. Could the volume of DMSO added have any effect in this situation?

We had performed the method described in "Identification of HSP90 inhibitors as a novel class of senolytics" using human endothelial cells and human fibroblasts. Briefly, cells were incubated with 10 uM of C12FDG (2h), then prior to analysis, DNA were stained using Hoechst dye 2 ug/mL and we observed green fluorescence stainning in both senescent and non-senescent cells. We also tried to change the C12FDG incubation times and concentrations and we obtained the same results. We are using another kit to detect SA-BGal (senescence detection Kit ab65351) as a control method and the results do not match. We have the attention of always preparing a fresh solution of C12FDG when starting a new experiment.  

We want to use this method for immuno fluorescent method to quantify senescence using INCell Analyser.

Do you have any suggestion that can help us?

acetocarmine used to stain DNA

My lab has recently be discovering a problem that some of our old nissl stain (2-3 years old) is turning to a disgusting orange/ brown color. Its not even every slide that was stained together, just a few every once in a while. Our slides are coverslipped with permount and glass and there's no air bubbles in any of the slides

Does anyone have any idea what could be causing this?

Hi everyone,

I am having some problem that my ICC staining is getting contaminated with fungus I suppose after adding the antibody with blocking solution which has Tween, PBS and Donkey serum. I checked one by one, when PBS alone no contamination, Donkey serum and PBS no contamination but all three together contamination is happening. Anyone knows how to troubleshoot this?

I stained HepG2 cells with propidium iodide (PI), calcein AM, and Hoechst. Dead cells were treated with PFA and Triton-X. The staining concentration and time were fine, but PI stained most of the cytoplasm in almost all cells, while Hoechst stained only the nuclei normally, indicating that the nuclei should be intact. I'm wondering if anyone has encountered this situation before and would appreciate any advice.

So I have been trying to standardise a zebrafish single cell suspension for downstream sequencing. I am consistently getting decent viability if I measure it via syto 9 and propidium iodide staining using a commercial single use hemocytometer.

But whenever I try to use the logos Luna cell counter or any other automated cell counter which uses 1:1 trypan blue (0.4%) staining, I am getting really poor results for the same exact samples. Any insight would be much appreciated.

Hi all, I have to postpone some stainings and imaging due to technical difficulties, but the cells are already in culture. They are hiPSC derived cortical neurons in 96well plates and the staining will be Bodipy.

- how long can I keep them in the fridge after PFA fixation?

- is it better to stain first and keep them that way or stain freshly? The structure I'd like to stain is lipid droplets, so I'm leaning towards the first possibility.

Thanks for all comments

I used to normalize my target protein using housekeeping genes. But now I have come to know about total protein normalization. So I would like to do the same. I want to know how to measure total protein using Ponceau S stain.

Hi everyone,

I want to do PI and DAPI dual staining for my cell culture.

Can anyone give me a protocol for this.

Thanks in advance.

Muscle frozen sections were stained with antibody against Laminin.

Hello,

I am attempting to stain 20um PFA fixed and frozen mouse brain tissues using the protocol below. This has been a very successful protocol for me. I perform side by side staining for anti-Iba1 and GFAP. The GFAP staining turned out well but the Iba1 continues to fail. Previously, I used anti Iba1 ( 1:500, Cat.# 016–20001, Wako) and it works well. This time, I switched to anti Iba1(1:500, Cat.# 019-19741 Wako) in order to increase dilution eventually but I started off using 1:500 as well. Using this antibody, no microglial were observed, but oddly GFAP was seen in the side by side tissue (smae tissue used for Iba1 staining) that was processed at the same time using all of the same reagents with the only difference being the primary antibody. I went back and tried the previous anti iba-1 i used and an additional one from neuromics and still not luck. Any suggestions as to how to circumvent this issue?

Thank you for your help in advance!

Kelly

Protocol:

Brain tissues were fixed in fresh 4% paraformaldehyde at 4oC for 48 h, followed by infiltration in 30% sucrose for 72 h. Tissues were then imbedded in cryoprotectant media (Cat.# 3801481, Leica, Deerfield, IL), flash frozen with dry ice, and sectioned using a cryostat (CM1950 Cryostat, Leica) to a thickness of 20 μm and preserved at -20oC in the cryoprotectant solution (40% PBS, 30% Ethylene glycol, 30% Glycerol). For immunohistochemistry staining, brain sections were blocked in 1% peroxidase (Cat.# 516813, Sigma-Aldrich) at room temperature (RT) for 10 min, washed twice with PBS, then further blocked in PBS blocking buffer (PBS-BB, 2% bovine serum albumin, 0.3% Triton-X-100, 0.2% non-fat milk) at RT for 1 h. PBS-BB was removed, and the sections were added to anti-GFAP (anti-rabbit, 1:1000, Cat. #7260, Abcam) or anti Iba-1 (anti-rabbit, 1:500, Cat.# 016–20001, Wako/ or Cat.# 019-19741 Wako ) antibodies that were diluted in 1XPBS or PBS-BB, incubated at 4oC overnight, and then probed with HRP-conjugated secondary antibody (goat anti-rabbit, 1:500, Cat.# 4050-05, Southern Biotech) at RT for 4 h. The sections were washed in PBS three times, mounted onto superfrost plus slides (Cat.#, 22-037-246,Fisherbrand, Pittsburg, PA), and briefly exposed to freshly prepared 3,3′-diaminobenzidine tetra hydrochloride substrate (DAB, Cat. # ab64238, Abcam) before dehydrating and preserving with Permount mounting media (Cat.# SP15-100, Daigger Scientific, Hamilton, NJ

I would like to use FAM [5(6)-carboxyfluorescein] (ex 493/emission 517) fluorochrome in a Immunofluorescence protocol and I need to fix the staining. I can't find any fixation protocol convenient for this fluorochrome.

Thank you in advance.

Hi all,

Has anyone ever performed a live/dead staining on THP-1 macrophages (M0) in a collagen gel? For my master's project I need to verify that my collagen gel is non-toxic to my cells. I seeded the cells in a 96 well plate suspended in 50 microL collagen gel.

I stained it with CTG and PI, but the microscope gave no signal/image.

Is there anyone who has done this before and could possibly share a protocol with me on this?

Tips are also welcome!

Thankyou in advance.

Hi,

I am using Acridine Orange staining to visualize autophagic cells. The staining should remain green in the nucleus (sometimes also in the cytoplasm) and turn orange within autophagic lysosomes, showing only orange dots in case of autophagy.

However, it seems that the orange coloring is the same and overlapping on the green one. Had this happened to anyone? How can I fixed the problem?

Also, I'm wondering if it is possible to fix with PFA 4% after staining.

Thank you.

Hi everyone,

I want to do PI and DAPI dual staining for my cell culture.

Can anyone give me a protocol for this.

Thanks in advance.

I need to know the best way to differenciate Th1/Th2 cells in human PBMC. Is it intracellular staining (for example IL-4/INFg produccing cells) or extraccellular staining (CXCR3,CCR4 etc). What is the most relayable and simple way? Im may opinion extracellular staining is faster and more convinient, but can i relay on tha data of such staining?

Dear community,

Recently, I'm doing an experiment to measure the permeability of endothelial cells(HUVEC, HPAEC). I understand that it is important to make sure that endothelial cells form monolayer before proceeding with Permeability Assay. So, I'm going to dye the cell membrane of the vascular endothelial cells and check it with a fluorescence microscope. I'd like to ask you what reagents do you use to dye your cell membranes? What I found in my paper are the ones I suggested below, and I'm asking if it's okay to use to dye endothelial cells.

1. Dil Stain (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate ('DiI'; DiIC18(3))) (cat# D3911)

2. CellBriteTM Orange Cytoplasmic Membrane (cat# 30022)

3. CellMask™ Plasma Membrane Stains (cat#C10046)

Thanks for your attention.

We´ve been using the Picro Sirius Red staining for several years now (done in out core facility) to show fibrosis development.

However suddenly our stainings, which usually had a yellow background turned orange, which makes the red fibers much harder to adequately quantify.

Our core facility hasn´t strayed from the usual protocol and no solutions were replaced or anything done differently than before, at least according to them.

We´ve done several stainings by hand, all of which have come out orange.

Does anyone have any idea how exactly this might have happened or how to make our background lighter yellowish as it was before?

The cell wall of Cryptosporidium and Cyclospora resemble Mycobacterium tuberculosis in consisting of mycolic acid ( low amount); therefore, the Modified Ziehl-Neelsen technique is a test of choice. Other biological stains like Rhudamin and Oramin Calcofluor white are additional options for demonstrating the oocysts of this group of protozoa.

I'm currently working with mouse peripheral blood, but several issues came out every time. Actually we are using as collection site the tail, blood is drip in eppendorf containing EDTA and inverted several times for better mixing. When the samples arrives in my lab, we use to gently invert again the eppendorf, for better distribution of anticoagulant, and 5ul of blood is placed on the slide. with another slide the smear is performed, the angle of smear is small, to have a thin smear and the velocity is low, to obtain a good distribution of blood all over the glass. Smears are air dried and stained with May-Grunwald-Giemsa by lab technician of Hematology dept. the next day.

The problems are related to frequent burst of WBCs and RBCs hemolysis, that occurs almost every time... but we cannot understand the causes.

Thanks for every suggestion

Please share the protocol

I need to extract cell nuclei from animal tissue before DAPI staining for flow cytometry analysis. I need to test a lot of samples, so I'm looking for a cheaper alternative to ready-made kits. Does anyone know a recipe for such a buffer?

I'm trying to detect intracellular iron levels in my cells in-vitro and stained using prussian blue. I fixed the cells in 4% paraformaldehyde, washed and let them sit in prussian blue mix from abcam for 30 minutes before washing and mounting them. When I checked the cells were stained a brownish color instead of blue. Has this happened to anyone before? Does anyone have any suggestions or know what's going on?

Hello,

I have an issue with my Alizarin Red staining. It gives random unspecific positivity stains throughout the tissue (Embedded in paraffin, dewaxed in Xylene). My question is, how do I remove these unspecific stains, or how can I remove the staining altogether?

Thank you.

We are establishing a stain-free gel protocol in our laboratory to normalize total protein and are having issues visualizing the proteins on the membrane after the transfer. We are loading whole cell lysate into 10% TGX stain free gel (Bio-Rad) and activating them using an Azure Biosystems C400 imager. Activation time is 5 minutes with UV302. After gel activation, the gel looks great in that the proteins can be seen after acquiring an image at UV302 set at auto exposure. Afterwards, the gel is immediately compiled with the turbo-transfer pack and transferred using the Trans-Blot turbo system (mini gel, TGX rapid 3min protocol, we've tried both nitrocellulose and PVDF). To my understanding, we should be able to visualize the bands on the membrane after the transfer and use this image for total protein normalization (I have also confirmed this with Bio-Rad technical support). However we cannot detect the proteins on the membrane using the UV302 auto exposure setting. (We have confirmed that the transfer was successful using other methods). I am hoping it is a simple setting issue with the Azure C400 imaging system however neither Bio-Rad nor Azure technical support can offer any additional support.

Also, according to the bulletin for the Bio-Rad imaging system, the protocol for visualizing the membrane after the transfer is "Stain-free Gel Application" protocol. What wavelength is this protocol?

Thank you for any support

Hello everyone,

We are trying to follow reactions involving PEG (2000 Da) by TLC, but so far we couldn't stain the starting material after running the plate. We tried iodine vapours, ethanolic sulfuric acid, phosphomolibdic acid and potassium permanganate with no success. Can anyone experienced with this task give us a hand?

Thank you in advance!