Cartilage - Science topic
A non-vascular form of connective tissue composed of CHONDROCYTES embedded in a matrix that includes CHONDROITIN SULFATE and various types of FIBRILLAR COLLAGEN. There are three major types: HYALINE CARTILAGE; FIBROCARTILAGE; and ELASTIC CARTILAGE.
Questions related to Cartilage
Hello every one
I Have some problems with tissue wash off when im trying to IHC. I tried some adhesive slides but I didnt get result. my sample is trachea with a lot of cartilages. has some body any advice to keep it more adherent.?
I am allowing the digestion of minced cartilage (post washing in PBS with 5% antibiotic) in type II collagenase (5mg/ml) enzyme for 7hours in continuous shaking mode at 37 degrees followed by filter through cell strainer, pelleting down cells and resuspension in complete media (10% DMEM) and plating in cell culture flask. I am getting cells suspended in the media next day, none of the cells are adhered to the surface. Please provide me suggestions to overcome this issue.
I need segmented ground truth images of knee bone, cartilage and miniscus from knee MRI images for my research work. Kindly inform me if anyone have it.
Recently many otolaryngologists are doing cartilage tymnoplasty particularly in children. I like to know about the ground experience of the researchers about the advantages and disadvantages of cartilage tymnoplasty. No hypothetical discussion please.
Hello everyone. I'm having a little conundrum in my lab. We've been running our h&e staining as usual and all of a sudden, slides present this "bluish" "smudged" area. It mostly occurs in glandular cells of colon, cartilage. We have tried troubleshooting it and:
- it is not the staining, as we had an external slide, and staining is perfect (colon)
- it is not a fixation problem as specimen are fixed long enough from 6hrs(small bxs) to 48hrs (resection post opening)
We are thinking it might be a processing issue, maybe the dehydration step, maybe a poor quality of alcohol (we use IPA).
Please see attached 2 bad staining from a colon bx and neck resection, and the external colon slide.
Hope someone could help.
I would like to do the alcian blue staining of tissue-engineered cartilage. For this, I have embedded my samples in paraffin and cut them into 10um thick sections. During the deparaffinization step (serial changes in xylene and ethanol), all my tissue sections are washed out from the slide. Does somebody know how to overcome this problem?
What is the physics and search stiffness measurements in industry however, do not forget that you are going use that technique on human tissue.
I have question regarding the use of Abaqus.
My goal is to make an extensive FEA of the pelvic region to investigate fracture patterns (big plans I know). To do this I would like to model the contact between the pelvis bone (E=17GPa), cartilage (E=10.25MPa) and the femur head (E=20GPa), this is where I struggle loads. In this model the hemipelvis will be fixed at the sacroiliac joint and the pubis. The loads will be applied via the Femur head.
So I decided to take a step back, to investigate the contact properties, therefor I've made a simplified the contact model (see attachment) by making a simplified box (named bot) with half a sphere (R=12), then a layer of cartilage (named kraakbeen) with outer R=12mm and inner R=10mm. and then the femur head is simplified as a sphere with R=10mm. The inner surface of the bone is tied with outer-surface of cartilage. The contact between the cartilage and the femur is supposed to be friction-less.
The FEA is done in 2 steps. The first step (step-contact) is displacement step, where the head of the femur is displaced 0.01mm to ensure contact. (displacement is placed with a BC)
The second step (step-load) is the step in which a load (same direction as the displacement) of 2500N is applied.
Preferably I would like to have the displacement-BC removed in the second step and only have a influence of the load that's applied on the femur. However my model does not solve that way.
If I continue the displacement BC into the second step, my model is able to fully solve, however the results are not correct. Since the stresses do not chance during the second step (in which 2500N) is applied. Meaning the application of the load doesnt seem to have any influence.
Can anyone help me how I correctly apply the load to the model and have proper boundary conditions of the pelvic-cartilage-femur?
Currently I use Leica BOND Plus slides to mount 5 micon thick FFPE sections of mouse knee joints. These have been fixed in PFA and decalcified in EDTA by experienced researchers, and have been embedded using the following programme in an automated tissue processor:
70% EtOH – 2h
95% EtOH – 2h
100% EtOH – 2h (x3)
Xylene – 2h (x3)
Histowax – 1h (x3)
I dry the sections overnight at 37-40 C on a drying rack, then bake at 65C for 3h and leave the sections to set at RT.
I dewax using the following protocol:
Xylene - 5 min (x3)
100% EtOH - 5 min (x2)
95% EtOH - 5 min
70% EtOH - 5 min
50% EtOH - 5 min
1x PBS - pause point until steamer reaches 99C
Then I proceed with antigen retrieval at 99C in a steamer:
dH2O - 10 sec dip
Tris-EDTA, pH 9.0 - 10 min
1x PBS - pause point at RT.
After the steamer steps, bone and cartilage seem to have fallen of the slide, folds are introduced, etc.
As for the other tissues, the muscle seems to have some folds, while the marrow is mostly intact and does not detach.
I've tried in parallel a different buffer (Citrate buffer, pH 6.0), an overnight 60C incubation in a water bath, as well as a pressure cooker. In all cases the same effect is observed, including if I just stick my tissues in water at 99C for 10 min. This leads me to believe the bone and cartilage tissue is not sticking properly to the slides.
Do you have any tips/recommendations on what I could be doing wrong?
Is there any database to identifies which protein isoform is expressed in a particular tissue?
I want to design primers for real-time PCR on cartilage samples. I need to be sure which mRNA variants are specific for cartilage.
I would like to visualise the cartilage wear on the surface of glenoid after friction test using a UV light (on macroscopic level). Could you please recommended me a florescent dye that I can use and won't affect the structural propertied of cartilage as I will be using the cartilage for histology after.
Thanks in advance.
I have developed ideas and concepts by which we can change the paradigm of OA to Cartilage attrition arthropathy, to facilitate a change in thinking, so that arthritis in the knee can be regarded as a condition that can and should be halted rather than being an inexorably progressive condition awaiting knee replacement.
please advice... as I would be performing histological staining using alcian blue Haematoxylin staining on zebrafish cryosections to look at cartilage element or strs.
I want to do cartilage staining on Xenopus embryos (st 45). Following the protocol, I tried to dissolve 4 mg alcian blue in 7 ml ethanol plus 3 ml acetic acid. I found alcian blue powder is very hard to completely dissolved in this solution. Is there any suggestions?
respectfully , i am ensiyeh kordparijaee , a student in medical biotechnology at babol university of medical sciences- from iran. i need the article by topic The role of stromal cell-derived factor 1 on cartilage development and disease , published in MAR 2021 at science direct written by Li,j ; chen,H.;Zhang,D. et al. for my research in the treatment of arthritis , please send it to me , if possible.the picture of its abstract has been attached
The dynamic mechanical analysis (DMA) test with frequency sweep is well known technique to characterize the viscoelastic properties.
I had gone through the different studies which had used DMA test to investigate the variation of storage and loss modulus with frequency for bone (compression mode) and soft tissue such as skin or cartilage (In tensile mode).
In literature it is reported that loss modulus decrease with frequency in bone but it increase for skin and cartilage.
I am currently modelling a load being applied in the joint zone of a hemipelvis, which correspond to a person while one leg standing.
Previously, I validated my model with an already existent one from the literature by focusing on the contact area and contact pressure parameters. Nevertheless, when it comes to comparing Von Mises stress between both models, the result variance has increased.
I have asked myself this variance question without yet confirming which is the cause.
Both models have the same boundary conditions and the same magnitude of load. Additionally, they also use the same elements and order of elements(C3D4).
After comparing displacement between them, it seems to be a slight difference (higher in the literature validated model) between them. I attribute this difference to the fact that the already validated model has a smaller thickness than mine in the zone of interest where the load is being transmitted..
Since von Mises stress is calculated from displacement in nodes, it seems plausible to me that the differences in stress between my constructed and the already validated model is due to thickness difference ergo displacement and stress.
Can somebody aid me on this matter?
- 36.13 KBCurrent model_Displacement frontal plane.JPG
- 38.18 KBCurrent model_Displacement lateral plane.JPG
- 42.90 KBCurrent model_Von Mises stress lateral plane.JPG
- 34.38 KBValidated model_Displacement frontal plane.JPG
- 36.06 KBValidated model_Displacement lateral plane.JPG
- 41.54 KBValidatedmodel_Von Mises stress lateral plane.JPG
I've been trying to isolate total RNA from guinea pig's knee cartilage. I've tried Qiagen RNeasy kit, miRNeasy kit, trizol method with lots of modifications and have also used the RNaqueous total RNA isolation kit from Thermo Scientific but I've not been able to achieve RNA ratio of more than 1.4 for cartilage and 1.6 for synovium. In addition to the integrity the RNA conc. is also very low because the amount of cartilage available from the knee of guinea pig is very less (<15mg).
I homogenise the samples using Bead beater (bead mill).
I need to quantify the glycosaminoglycans from articular cartilage.
I saw some protocols using the DMMB assay. Is there a similar protocol of quantification, but using alcian blue instead of DMMB?
I am working with cartilages that are in contact on the hip. In order to obtain a contour plot of the contact pressure I performed a mesh sensitivity test by selecting linear tetrahedral elements (C3D4). Nevertheless, I was advised to use a finer mesh for visualizing von Mises stress.
After doing some reading, I came across the term "reduction integration scheme" which states that strain and stress in FE uses an integration point in the center of the element. Thus, since Contact Pressure is measured in nodes of elements, it makes sense to me to increase the density of the mesh to obtain better results.
Am I correct? or Is there another explanation for this?
I’m implementing a relatively highly cited academic research paper: “Interobserver
Reproducibility of Quantitative Cartilage Measurements: Comparison of B-Spline
Snakes and Manual Segmentation”. In short, the paper outlines how you can get a
B-spline to outline knee cartilage in a 2D slice of an MRI. I am stuck at minimizing the
cost function using gradient descent. I do not know how to gracefully apply a partial derivative to a line integral where the line is a B-spline contour in a 2D image. A full explanation of the problem and my current solution is attached, however I make a poor assumption to get to that solution. If anyone could double check my math or has any ideas I would greatly appreciate it.
Hi all, I'm searching for volumes of synovial fluid in sheep osteoarthritis (or cartilage defect) model? Any estimation through experience, publications would help. If you have references to other large animal model or human patients, that would also be helpful.
Many thanks in advanced!
I am using this protocol (https://www.scientistsolutions.com/forum/cytology-histology-and-ihc-organelle-and-cytoskeletal-staining/cartilage-staining) to do cartilage staining on organoids that have been fixed in 4% PFA, dehydrated in 10,20,30% sucrose+sodium azide, frozen in tissue freezing medium, sectioned on a cryostat and kept at -30*.
Would I need to take any additional steps before re-hydrating the sections and next staining with the hematoxylin?
Thank you in advance!
Dearest hive mind,
I have been trying to get a staining going for collagen type II in human specimens (de-calcified). Until now, without great results. My initial approach has been with enzymatic antigen retrieval with pronase and hyaluronidase according to Amirtham et al. 2019 (1 mg/mL Pronase for 30 min. and 5 mg/mL Hyaluronidase also for 30 mins). I had a suspicion that the enzymatic treatment would be quite rough on my sections (formalin fixed, paraffin embedded), so I did not do 37 degree during incubation with the enzyme. Of course the sections basically fell of (I guess the enzymatic treatment was too harsh). But, the most annoying thing was, that the tissue that was still on the slide, did not stain nicely (bone with clear areas of cartilage (Specimens were demineralized in 10% EDTA ). I used Dako Envision and AEC as chromogen (In my old lab we used DAB so this is a first time for me). The antibody is a polyclonal rabbit anti human Collagen II from Abcam. The incubation time at room temp with the primary antibody was 2 hours. Do any of you genious-researchers have any good ideas about what I am doing wrong. I'm used to looking at histochemistry, and boy, with a bit of Picrosirus Red you are not in doubt about what is collagen and what is not - but maybe I should not expect to see an intense staining. What I am ultimately trying to do, is to show where there is Collagen I and where there is collagen II as a way of saying "this is cartilage as opposed bone" - am I approaching this from a wrong angle?
I am trying to stain bone tissues from human knees however I am having a hard time with antigen retrieval. I use citrate buffer and a water bath set at 85 ° for 20 minutes and I leave the tissue in the solution until it reaches room temperature. I still have some tissue left but they become so fragile that they come off after a few washes during blocking step.
I do use positive charged slides too.
Can anyone suggest a good method for unmasking antigens when using bone and/or cartilage?
I have followed the advice on this site, Penn State research papers and numerous www.ncbi.nim.nih.gov articles. A man was diagnosed with osteoarthritis of the knee due to a severe motocross accident. I've attached his x-rays from before and after the plates were finally removed from his leg which was one year after his crash. I need advice. Does the second set of x-rays reveal a total healing, or are we just seeing the effects of numerous chondroprotective supplements which were given to him in the hopes of opening the space in his knee? I ask because it seems to soon to experience this level of healing. I've also attached the patient's supplement list with dosages and dates. Please understand that the supplement list was created for my benefit and not necessarily to be read by others. By this, I mean I was only attempting to keep my research straight.
I have to paraffin embed shavings of cartilage ...super small less than 1X1 mm in size for H&E staining. I will have a scoop of these shavings that will be glued together during culture using Evicel. After 4 weeks in culture I'll have to pick up the blob of glue with the cartilage and proceed for histology. Any pointers as to: duration of fixing in 4%PF - dehydration - embedding and sectioning? I will have two groups with and without glue. The scoop of cartilage without glue will be a challenge as the tiny pieces will be all over -floating around in media.Thanks-Anu
We are making a 3D mechanical model of the thorax and we need the properties of the materials therein. The density of bone is about twice that of water, and I found unreliable affirmations that cartilage is less dense than bone. Does anyone have more reliable information? We are interested in costal cartilage, but would be satisfied with any old type of cartilage for initial, order-of-magnitude simulations.
Tendons, ligaments, cartilage are composed of collagen. There are articles saying that collagen supplementation can help with tendon injuries. Where is the scientific evidence to support these claims? How is ingestion of collagen linked to an increase in collagen synthesis in the body?
I'm doing AP IHC of collagen I and II on cartilage frozen sections.
The results look quite different than on paraffin sections. I tried different fixations methods (formalin, EtOH) and the antibodies should work on frozen sections according to the datasheet. It seems as if the collagen that is in the lower part of the cartilage (closer to the bone, higher proteoglycan content) does not stain as it should. Biggest difference between the paraffin and frozen sections is the lack of antigen retrieval in the protocol for frozen sections. Could it be that I still need to do antigen retrieval on the frozen sections?
protocol in short:
frozen sections, non fixated
- fixate in 3.7% formalin for 10 minutes
- wash in PBS-T
- block with 1% BSA/5%HS in PBS-T 30 minutes
- primary AB @ 4 degrees overnight
- wash with PBS-T
- secondary antibody conjugated with biotin 1 hour
- wash with TBS-T
- ABC-AP complex 1 hour
- wash in TBS
- incubate with AP substrate solution 15 minutes
- stop reaction by washing in TBS
Thanks for your help!
I'm having trouble with facial cartilage staining in zebrafish (120hpf).
I followed this protocol: https://wiki.zfin.org/pages/viewpage.action?pageId=13107315
except that I had fixed in PFA overnight at 4 degrees, and had to bleach for much longer (a few hours).
I followed the protocol through but saw no staining!
There are a few issues that I think could be the problem.
This protocol makes up alcian blue at pH7.5 but I have seen others which state that it is active at pH 2.5... Any experience with this? I've seen that at different pHs it seems to stain different things though, so not sure what is best for facial cartilage then?
Second, my alcian blue (0.04%) appeared to have precipitated, and appeared to go back into solution upon shaking - but maybe it didn't really and requires heat? Or another step to avoid precipitation?
Did I over-fix or over-bleach?
Any help would be much appreciated!
Has anybody ever used azole antifungals (fluconazole etc) in their cell/tissue culture? I work with primary human bone/cartilage tissue and I struggle with amphotericin B resistant fungal infections that most likely come from the surgery theatre. Are there any alternative antimycotics that would be safe for the cultured cells?
Thanks in advance,
I work with congenital disorders of bone and cartilage. I send my proband samples for exome sequencing and validate the variant by doing sangers in lab. Recently I got a result with heterozygous duplication inframe (c.2026-1_2042dup p.Ala680_Gly685) I am facing a difficultly in capturing these variants by sangers sequencing! How do you design a primer for inframe duplication of 18bp with 6 residue change?
I am actually using bat embryos, but they should behave similarly to mouse or other mammals. We have tried the staining before, but to no success. We think that the PFA may have created a barrier at the skin which prevents Alcian blue from entering the sample, because the stain works perfectly when it is sectioned.
I need to ship some mouse long bones and I don't know how they should be stored since I want to analyse the bone and the cartilage after the shipment. I've seen that I can stored them with dry ice but I haven't found a specific procedure.
Thank you in advance.
I am interested to collaborate with any researcher with suitable animal model for osteoarthritis. I have a new therapeutic approach for repairing damaged cartilag that requires serious investigation. Please get in touch, my e mail: email@example.com
we face many patients, females, young and complaining from cartilage softening and only known while doing knee arthroscopy .
the alignment angles was within accepted range.
what to do to eradicate this lesions
I am trying to do indentation test for my cartilage sample, but I am curious the boundary between compression and indentation, (like the image shows). I was wondering is there any standard use to define the size relationship between indenter and sample?
If I already know my indenter size is 3 mm diameter, what is the minimum size for my tested sample?
May I use FEA to check the deformation at sample edge, if the indentation process do not bring any deformation to the edge which means it is indentation process.
Osteoarthritis is characterised as the deterioration of the articular cartilage which then causes bone-to-bone contact. Glucosamine is a primary constituent of cartilage proteoglycans, which is why there are claims that glucosamine could possibly be an anti-arthritic. Despite this, there has been no current and impressive evidence supporting this claim. Has there been any current advances on this claim and if so, what are they?
I'm from Argentina and i'm working whit genetic expression of degenerative joint disease in dogs. So we're trying to isolate RNA from cartilage to further NGS analysis. But till now we haven't get good results. We need some advices about which is the best kit or method to isolate RNA from cartilage tissue. Could you tell me some about it please?
My best regards
I am trying to detect senescent cells in OA cartilage. For this I have tried SA b gal staining but the results are not good with very little staining . Since enzyme activity is very tricky to detect and fixation and freeze thawing has adverse effect, I am stuck with the experiment. I am not sure whether doing immunostaining with b gal antibody would make any difference. Fluorescence substrates may be another option as well. persons with first hand experience with these please give your opinion. Thanks
Do you prefer to use cartilage for reconstruction or bony plate ?
Any available literature regarding this?
Cartilage has neither blood supply nor innervation. However, patients with cartilage lesions typically present with pain. The question does not aim at advanced osteoarthritis, which is associated with inflammation and expression of pain-mediating cytokines. The main emphasis is the focal damage after the acute phase.
Who plays the predominant role, mediators or biomechanics?
I would like to obtain mitotic figures of chondrocytes, so I am staining cartilage tissue embedded in paraffin with crystal violet. I would be very thankful if anyone kindly shares his protocol or experience.
Also, there are some confusing terminology between crystal violet and cresyl violet in the literature sometimes. Are they totally different or share common characteristics/related structures?
Thank you in advance for your help.
I have a lipid emulsion for topical application that I want to test on the cartilage cells. I wanted to know if the different components of the emulsion (oil and surfactant) can cross all the barriers of the skin and the muscles to reach the cartilage?
I need publications or references please .
I'm using two different types of PI ( Photoinitiators )
VA-086 and IRGCURE 2959 to Photo-curable my polymers (methacrylated Alginate and methacrylated Gelatin) .
- Could you please suggest the proper concentration of each PI to form hydrogel (Bioink) for Cartilage applications?
- what is the proper UV intensity (xx mW/Cm^2) that I can use for less time please ?
I have tried to stain rabbit cartilage using different hematoxylin stains (Harris and Mayer (very old one)). However, I can not see the nucleus clearly.
The tissue was fixed 2 days in formalin after it was decalcification and embedded in paraffin for 5 days. After dewaxing, the slide was stained with hematoxiline for 16 min.
Would you please let me know if you have any proper protocol for cartilage H&E staining?
Can someone advice on a protocol to detach MSCs from the surface of scaffolds for PCR analysis.
I am working with decellularised trachea scaffolds ( Pig) seeded with MSCs. Cells only attach at the surface. I want to avoid grinding the tissue because it contains genomic DNA remanants and dense ECM cartilage. and I got very low yeild.
We found that sliding of cartilage against an artificial meniscus could increase friction during the swing phase and long term elevated friction could cause cartilage wear.
I am doing IHC with bone and cartilage samples (eg. growth plate).
For antigen retrieval, I usually heat the samples in Citrate buffer (pH 6.0) using pressure cooker (for 1 min at max. temp and pressure and cool down for 20 min.)
But, bone and cartilage part are peeled off from the slide in high temperature.
In case of proteinase K - induced antigen retrieval, tissue is okay, but it doesn't work, no staining.
Any better antigen retrieval method for bone or cartilage sample?
I have tried to take a part of the vertebral column and dissolve the surrounding tissue with 6% potassium hydroxide but after one day I found that the cartilage has broke into very small pieces. do you have any tips on determining ray fish age?
Hello I am performing the chondrogenic differentiation of adipose-derived stem cells in bidimensional cultures.
I would like to ask which one is the best protocol for the staining and the semi-quantification by absorbance at 630 nm.
I read several protocols... I have Alcian Blue 8GX (powder) from sigma (A5268). I saw in short, two kind of staining with Alcian blue in Acetic acid (pH - 2.5) and Alcian blue in 0.1 M HCl overnight after 20 min of fixation in 95% methanol (also I have doubts with the fixation... formalin, PFA 4%...?). The second one seems to be the best for cell layers in 2D, however I am a little afraid of the 0.1 M HCl (Did anybody tried it?).
Finally for the extraction of Alcian blue in order to make the quantification I read 6h of incubation in Guanidine-HCl 6M, correct?
Thanks in advanced,
Someone here recently posted a question about counterstaining Oil-Red-O.
I have a similar question. Nuclear staining with Hematoxylin would be ok, but are there other stains (that don't use ethanol) that I could try? I was hoping for something purple or blue that I could use to stain cartilage or cell membranes. I am using the Oil-Red-O to stain adipocytes so cell membranes are not as critical (the red cytoplasm is what will inform me).
I am currently having problems with IHC(immunohistochemistry) of a nuclear protein. I am working with human osteoarthritis cartilage tissue(damaged region-undamaged region) and mouse cartilage tissue(DMM-SHAM and young-aged). I am using peroxidase-based methods while blocking endogenous peroxidase with 0.3% H2O2 for 10 minutes. I also have a negative IgG control(there is non-specific staining here as well). I used both the trypsin and heat antigen retrieval method(they both have the same problem), blocked the samples with 1% BSA in PBS for 15 minutes. Also, I added a permeabilization step because I am working with a nuclear protein. The problem is that there is non-specific binding and high background where it is not supposed to have a signal and no signal where there should be staining according to my hypothesis. Can anybody help me with this problem?
I am currently having problems with IHC(immunohistochemistry). I am working with human osteoarthritis cartilage tissue(damaged region-undamaged region) and mouse cartilage tissue(DMM-SHAM). The problem is that there is non-specific binding and high background where it is not supposed to have a signal and no signal where there should be staining according to my hypothesis. Can anybody help me with this problem?
I have a sample of human articular cartilage that has been freeze-dried last year and kept in a freezer until today. I'm not sure that it can be used for a new experiment because of the possibility of changes in its biological and mechanical properties. I'd be so grateful if anyone who experienced the same situation or has the knowledge, shares his/her information with me.
P.S.: Is there any test or characterization method that can validate the consistency of the mentioned sample properties?
Hi. I am currently working with new materials that should be tested on a scarified skin.
I am employing the widely-accepted wound model using the ventral surface of a rabbit ear, which was originally suggested by professor Mustoe. In brief, this model involves full thickness excision of rabbit ear skin and perichondrium, leaving the bare cartilage to heal itself and form a scar wound.
However, i am having some drawbacks. From some samples, the hypertrophy of the rabbit ear cartilage, rather than the dermal portion, is too prominent. This makes the analysis of the dermal portion very difficult.
My guess is that there had been some random microdamage to the cartilage portion during the removal of the perichondrium, but that leaves me no choice but to leave the perichondrium, which will accelerate the healing and make the wound UN-hypertrophic.
Is there a way to maximally prevent cartilage hypertrophy, but only achieve dermal hypertrophy using this model? Thank You.
I am trying to do a 3D cell culture for MSC cells and condrocyte Cell lines for cartilage application using different concentration of photocrosslinking alginate and/ or gelatin hydrogels?
I was wondering if anyone can refer me to a step-wise protocol for culturing the cells using those hydrogels please?
I need suggestions for how to properly homegenize cartilage tendon. Right now, I use a pulverizer dipped in liquid nitrogen and then take the powerderd pulverized sample and put it in a RIPA buffer. I have tried multiple types of RIPA buffers and all leave at least some amount of sample out of the solution. Usually, even if the sample is completely powder, after using a motorized homogenizer most of the parts congeal back together. I am working in a lab where mostly muscle homogenizations occur, and no one understands why my samples re-congeal into a ball after pulverization. I think the fibrous tissues are hard to break down and want to stick back together, but I am unsure of how to make them stay in solution. I tried a mortar and pestle over dry ice once, and the same thing happened- actually it was worse than when I pulverized it into a powder. Maybe I should find a different RIPA recipe(?) but I have already tried a couple I made and one I bought and it's not a whole lot of diffrence.
RIPA recipe 1, 50 mL: .302 g Tris, .48 g NaCl, 0.05 g SDS, 0.25 g Sodium deoxycholate, 0.5 mL Triton X 100, pH 7.4
RIPA recipe 2: 100 mL: .242 g Tris, .8775 g NaCl, 0.29 g EDTA, 2% SDS, 1% Triton, pH 7.5
*** RIPA recipe 2 has worked the best so far
RIPA from Sigma-Aldrich:150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0
This is the image of Total RNA non- denaturing gel electrophoresis. I performed a total RNA extraction from dog (cartilage, blood, synovial fluid and knee ligament) tissue. Can someone please tell me what are the two bands intercalated between the 28s and 18s bands showing in the 3rd lane? Thanks
After digesting cartilage tissue in a collagenase solution, I always observe tiny oil-like droplets floating the digested suspension. Has anyone made a similar observation? Is the tissue a source of the oil droplets or is it the collagenase?