Brain Dissection - Science topic

Hi, Has anyone done dissection (e.g.,brain dissection) using the laser capture microdissection, and done the qPCR analysis on the samples? How was the RNA quality and which kit did you use?

the recent researches have shown that blind people react(smile) to the smile of a person in front of them. how this connection occures?

I would like to take rat kidney tissue and isolate from that the proximal tubule cells for immediate use in a Clark-type oxygen electrode to study mitochondrial respiration. I do not want to study isolated mitochondria (for reasons I will not go into here), and using tissue sections (e.g. 200 micron thin slices) does not allow full respirometry studies to be performed, only crude O2 consumption rates (even with permeabilisation).

I have seen lots of protocols for isolating PTCs for use then as primary cell lines however I will not be using the cells for this purpose. In my experiments the cells will be discarded after the short respirometry studies have been completed. I'm unsure of how much of these protocols I should follow if I just want to obtain a cell suspension for immediate use and not prolonged culture. Ideally I would like something quicker and simpler than most of the protocols I have found so far seem to be.

Prior to respirometry studies the naive kidney tissue will have been sliced to 200 um thick and exposed to certain insults (e.g. LPS, septic serum, etc.) for around 90 min. I would like a method that is as quick as possible to get the PTCs from the tissue in a state in which I can add them to the Clark electrode. This is in order to observe any acute changes that may have occurred to the mitochondrial function during incubation which may also revert relatively quickly once removed from the insulting environment. Once I have a cell suspension I intend to permeabilise to allow addition of the respiratory substrates, ADP, uncouplers, etc. required for respirometry studies.

If it's likely that this potential method will encounter too many problems I may first isolate the PTCs and then expose them to my various insults immediately before washing and beginning the respirometry studies. In that case would I need to culture the cells, or could I just expose them in suspension? Ideally I would however like to be able to expose slices to the insult (for reasons of consistency with other methodologies I am currently employing) as initially suggested.

Many thanks.

I want to do rat brain immunohistochemistry, and I find that rats were perfused transcardially with PFA under deep anesthesia in all published papers. But now some people in pathology told me that rats can be decapitated and immediaterly to get brain, after saline washing, the brain fixed to 4% PFA is OK. Should I to do perfusion at first?

Hi, we got 15mm x 15 mm x 0.15 mm Mica substrates. But after cutting by scissors, we found many cracks, as shown in the image. Does anyone know how to cut it gently without introducing those cracks? Thank you very much.

I have to isolate microglia from brain tissue for my research. Almost every protocol about microglia isolation suggest a perfusion with ice-cold PBS/HBSS .

I want to know why perfusion is necessary before brain dissection.

Thank you.

I am worried of its DNA, that it may be damaged....

I am currently performing a craniotomy/durotomy procedure on rats (Long Evans – Hooded Blue Spruce 450-600g) to expose brain tissue for optical coherence tomography. To open the skull, I am using an engraving bit (Dremel size 105 & 106) to cut a 4mm x 4mm area of the skull. Once dura mater is exposed I am able to lift the skull off cleanly without damaging the brain tissue. The dura mater is then carefully lifted with a tweezers and cut with a micro scissors. During roughly half of the procedures the underlying meningeal layers are removed cleanly along with the dura. However, if the pia does not come off cleanly (typically when it contains large perforating cortical vessels), any attempts to remove it results in significant bleeding. This obviously inhibits the imaging process by blocking the light aimed to penetrate tissue. Occasionally the skull bleeds significantly as well. Thus, if I am unable to avoid excessive bleeding, does anyone know of any non-pharmacological methods for addressing this problem (other than Gelfoam Sponge or Actcel hemostatic gauze)? Or any pharmacological methods that would not compromise hemodynamics? In addition, the exposed brain tissue becomes inflamed, varying the focal length of the lens and further distorting the images. What is the best way to control for this inflammation? I have been applying aCSF (stored in an ice bath) to control for this. I have also tried opening another area of the skull (left lateral the sagittal suture, posterior to bregma and anterior to lamda) to allow for a release of some pressure. So far, this doesn’t appear to be beneficial. Lastly, what is the ideal dosage of Isoflurane to administer a rat under hypoxia and/or hypercapnia? After collecting baseline data (at normoxia), the rats are exposed to a hypoxic/hypercapnic gas mixture (10% O2, 5% CO2, 85% N) in order to examine capillary dilation and perfusion of the cerebrovascular reserve. Some of the rats remain stable under this condition, others expire within a few short minutes. Does anyone have an explanation for the variability? The rats are all the same breed, age, and are close to the same weight.

My goal is to examine hemodynamic activity in real-time. Therefore, any solutions to these issues should not further compromise normal blood flow, or vascular restriction and dilation.

I am trying to hole punch for RNAseq in specific subregions in the rat brain. In order to be more accurate I want to do 300 micrometer. I am wondering if I should buy a tissue slicer.

Hello, 

I have a problem with the cryoprotection of mouse hippocampal slices. The samples are PFA-fixated and cryoprotected in 30 % sucrose in PBS solution; they would normally sink to the bottom of their wells in the 96-well plate after a couple of days in the cryoprotectant, but this time none of the samples (there are five of them all in all) would sink. I have already encountered such a problem a couple of times before, and according to my experience it is not a good idea to proceed with samples that are still floating, as that implies that they have not been cryoprotected well enough and therefore are very likely to get damaged during sectioning.

I placed them on a shaker in a cold room now, as I hope that that would help the cryoprotectant to enter the samples; however, I am not sure that that would work. Therefore, any advice would be very appreciated. 

Thanks a lot in advance! 

Max

I work with Rat & Mouse brain tissue. I have hippocampal slices in a 6-well dish that I have fixed. We keep stored at 4 degrees. We have changed the fix to PBS for long term storage and parafilmed the plate for spillage. However, I noticed that I had a slimy mold (look like a tadpole swimming) in one of my wells. Worried about contamination what's the best solution to preserve brain tissue without damaging the tissue or contaminating it?

I am Working on Primary Neural Cell culture from SVZ of the Postnatal Mice Can anybody Help me to Provide Some guidance how to Precisely cut this part. and How to recognize SVZ in Brain of Postnatal mice. If you have some Videos Illustrative Diagram I could be better for me?

I use 50ug/ ml Gentamycin in my culture media already. I have to dissect the sub-ventricular zone under a microscope which takes a while to do and all the steps upto the collection of region of interest are performed outside the hood. Please suggest if I should add the antibiotic in my dissection medium as well, for extra protection from the contamination.

How can i cut slices of cerebellum in a good way? I think someone who studies neuroscience. 

I need to dissect these nuclei of macroscopic manner.

I dissect the animal, intracardially perfuse with 0.9% Saline, then with few ml of 4% PFA intracardially. Then i remove the brain and fix it in 10 % formalin for 2-3 days after which i embed the brain in paraffin and process it. During paraffin sectioning, i consistently observed there is vaccum in hippocampal area, which is like a shrunken hippocampus. I could literally see a cavity while sectioning in a microtome.  Although i can get hippocampal sections, little beneath the actual area.. Can anyone suggest any reason for this shrunken/Vaccum, which i encounter in hippocampal region? Does this cavity arise due to the Circle of Willus, which directly supply hippocampus, which is situated just above the basal midbrain????

I have flash frozen whole honey bees, and would like to dissect out antennal lobes and mushroom bodies for gene expression work. Is the ICE reliably good for preserving RNA in small bits of tissue? Also, does it mess with structural integrity of the tissue (ie turning it to un-dissectable mush)? 

In term of solutions to use during or after dissection, dissection conditions, drugs to use, etc.

Apparently, mossy cells are very fragile/sensitive. How can I improve their survival rate to be able to record them later?

Dear friends, I need to use tungsten needles to deliver dextran-conjugated fluorescent die to the spinal cord of Xenopus tadpoles. I plan to label some reticulospinal neurons retrogradely. I've never worked with these needles, and so cannot quiet imagine them, both in terms of how they look, and in terms of how they would feel during the dissection. FST have 3 types of needles in stock: 0.5, 0.25 and 0.125 mm in diameter (link below). Which one would you have used for this task?

Educated guesses with any kind of rationalization are very welcome, as my best other option is to order the middle one just because it's in the middle.

The link to the FST site:

I would like to study neurosecretory cells of the brain of ceratitis. Can I use the carnoy as a fixative directly on head without dissection of the brain?

Can anyone definitively answer the question: do fixed brain samples in PBS with sodium azide need to be refrigerated for long-term storage?

I want to dissect visual cortex tissue for quantitative RT-PCR and I found that it is difficult for me to get visual cortex tissue evenly from diffrent mice. Does anyone have any suggestion? Any advice is welcome. Thanks a lot.

Can anyone help me in dissection of rat hippocampus and amygdala ? if possible with dissecting manual of rat brain?

As of late, I am having trouble picking any cells from brain tissue slices using immuno-LCM techniques. We have successfully picked single and pools of single cells from brain tissue up until recently.  Now it seems that we are consistently unable to pick up any cells. Nothing significant has changed with respect to the equipment, protocol, or reagents (to my knowledge).  I have been troubleshooting for a while now and am nowhere closer to any sort of solution for this mystery. We use the following protocol for tissue sectioning and immuno-staining:

slice OCT embedded brain tissue using cryostat at -20C (10um thick slices)

store slices on charged slides (for better adhesion)

store sections at -80C until staining and picking

we fix slides using ice-cold acetone (1min)

air-dry slides post fixation (~ 2-3 min)

block with 3% BSA solution

rinse with 1X PBS

add primary Ab solution (incubate for 3 min at room T)

rinse with 1X PBS

add secondary Ab solution (incubate for 3 min at room T)

rinse with 1X PBS

dehydrate in a series of EtOH solutions of increasing conc. (70%, 75%, 95% 100%, 200%)

final dehydration in xylene (100% 1 min, 100% 4 min)

air dry and store in 50mL tube with dessicant for 10-20 min

I have tested a variety of factors targeting various aspects our protocol:

longer acetone fixation times (5-10min), room T acetone (2min), longer EtOH baths (from 30s to 2 min), longer xylene baths (5min-10min), used new reagents (unopened bottles), new caps, uncharged slides, longer dessicant drying times (as long as 45 min), and have adjusted environmental conditions as best as I can in the LCM room (relative humidity ~mid-30%, which we have been able to pick cells successfully in the past). The IR-laser is able to wet the caps fine as well.  We are able to pick up cells from older tissues that have been used in the past, so we do not suspect an issue with the LCM itself. 

At this point, my colleagues and I believe it has something to do with the dehydration of the tissue (we note that in tissues that yield successful microdissection, they are typically opaque and white upon fixation and/or xylene dehydration).  However tissue that prove difficult to pick from are not nearly as opaque (more a translucent white - faded).  I would appreciate any insight from the community as to how we can improve consistency in tissue dehydration and/or techniques that will improve success of tissue microdissection. Any insights or thoughts would be helpful, thank you! 

I want to extract RNA from Fly larval brains. I had problems of degradation before. I am planning to use RNA later this time. Is it advisable to use RNA later while dissecting the brains or dissect the brains in water and transfer them to RNA later. Please provide me the details?

I want to micro dissect the brain of rats and mice to isolate hippocampus, prefrontal cortex and other brain parts. Can help me out to find the best video on micro dissection of brain either in youtube or any other website? Thank you.

I am trying to collect the hippocampus, prefrontal cortex and amygdala from adolescent rat brains (unfixed).  Is there a way I could validate the accuracy of the dissection to rule out contamination from adjacent brain regions? For instance, I have been searching for molecular markers of the hippocampus that are absent in adjacent regions, and vice versa.

Thank you

I will be removing whole mouse brains to be later used. So, I need to know the best methods to store the brain.

I always end up poking the brain to get rid of the vessels and butcher the surface.

This is proving to be too difficult. Any help would be appreciated.