Hi, we normally deal with fixation of brains by different methods, sectioning, staining of embryonic brains from mice. However we are mainly interested in cortical development. But never dealt with hypothalamus. However i assume if you are doing immunostaining then our protocols should also work. I can share them if you need.

Please can you share with me the protocol?

I believe that have not difference between the methodology to analyze cortex and hypothalamus.

Here you go:

1. Fix the brain after removal in 4% PFA for cryo). If its an adult brain then fixation should be overnight at 4 degrees on a shaker, if its embryonic till P0 then 2 hours is enough. Wash once with PBS and cryo protect it in 20% sucrose (in PBS) overnight till the brain sinks at the bottom of the tube. You can not put the brains in Tissue tek( special for cryo) and then make 10 micron- 15 microns cryo sections.

For enhanced signal by antibodies, you could use antigen retreival by immersing the slides in 10mM sodium citrate (with 0.5% Tween 20) and boil it in a microwave for 10 min. wash in PBS once it cools down ...subsequently you should block and then use it for immuno stainings.

I need to know what age of mice you are using..coz other protocols might not work with very old mice brains..like fixation with carnoys reagent and embedding them in paraffin.

if you need more details then do let me know..

Many thanks Bhavin

I am using C57BL / 6 mice with approximately 4 months of age.

You also do fixation by perfusion or by immersion only?

I'll do some tests with your protocol and then I'll show you the results!

Thank you very, very much.

I use immersion, but since 4month old brain is big, I would suggest you to separate the two hemispheres and fix it over night

Hi Aaron,

I am very grateful for your answer! Thank you very much for spending your time with the details in the protocol. It helped me a lot since I am starting now my phD course and I am working with something very different from my master project.

I have some doubts and hope you could help me again!

I have here the Peristaltic Pump Gilson 3 to perfuse. I don't have much experience with perfusion, so I would like to ask you which flow rate you use in your perfusion and how long approximately does it last.

In the item 5, in which you said that the serum should match the secondary antibody: We have here just the bovine one (BSA) and my secondary antibodys vary (I have rabbit, gota antibodys)..so I would like to have your oppinion, if you think it is not gonna work.

You use SuperFrost slides that are precoated with gelatin. We have here just silanized slides. Do you think this can work or would it be better with gelatin?

In the item n 6, do you do this 3 PBS washes and antibody incubations in a well plate or do you do it directly in the slides?

Thank you again,

Andre

Hello Andre,

I use basically the same protocol as Aaron, but I fast-freeze muy brain slices (we cut a slice that cointains the hypothalamus using a mouse brain-mold) in gelatin. I find it much simpler to cut in the cryostat later with gelatin than tissue tek, for example. That of course, if what you need are thin slices. You can always cut with a vibratome too, if you need to screen a larger portion of the hypothalamus.

We mount the cuts in SuperFrost Plus Glass, and I've found they work fine even for in situ procedures (where you heat the slices to 72ºC). They work perfectly fine for inmunostaining.

The bloking steps and such always depend on the primary antibody. Here, we use mostly NGS, except for goat primary antibodies and it usually works just fine. We don't block prior to adding the primary antibody but instead, we add the NGS in the primary antibody mix (2% NGS - 0,3%TritonX-100/KPBS-antibody) and incubate ON at 4ºC.

We also fix our brains ON in 4% PFA every time, even if the perfusion was correct. We found that most antibodies work, but then again, if you have an antibody that needs a little fine tuning you'll have to try different protocols.

Best of lucks!

Dani

Daniela and Aaron, thanks for the answers!

Before yesterday I did the infusion of 2 animals, I am increasing the concentration of sucrose (10, 20 and 30%, overnight). Tonight I'll include their brains in OCT and tomorrow I'll cut in a cryostat.

It was the first time I did perfusion, so tomorrow I'll make a histochemestry to verify the integrity of the tissue. If the tissue is intact, I'll start with IF.

Daniela, you use NGS independent of antibody or do you use NGS only for antigoat secundar antibody?

I just know that mold of mouse brain, very interesting. How do you do this gelatin? Even using gelatin, you keep your material in the freezer?

Tomorrow I'll tell you the news!

Thank you again,

André

Hello Andre,

Not mouse brain, but rat brain. Perfusion-fixed with 4% paraformaldehyde and then cut at 50-100 microns on a vibratome works well for immunocytochemistry and in situ hybridisation (you may need antigen retreival for some antibodies to work). For the confocal you need something that fluoresces e.g. the antibody or biotinylated nucleotide detection system. A wonderful general stain for the confocal that gives an appearance like Cresyl violet or other 'Nissl' stains is YOYO-1 iodide. Here are some papers using the CLSM on rat brainstem. Remember to coverslip using a water based mountant. Avoid glutaraldehyde in the fixative as it ruins immunostaining and gives background autofluorescence. If I were sectioning for the hypothalamus, I'd cut a block of fixed brainto include both thalami and adjacent cerebral cortex and brainstem and section serially at 100 microns on a Vibratome (should take about 30 mins. Collect every section in a multiwell dish in buffer. Then place the multiwell dish on a dissecting microscope (or an ordinary on if you can rack the stage down far enough). You will see a lot of detail and will be able to pick out the third ventricle, thalamus and the hypothalamus under it. If you start in the middle of the series where the hypothalamus is unmistakeable you can move to sections either side of this and keepgoing until you can see the hypothalamus. This is the quickest way of defining the rostrocaudal extent of a brain structure. I usually do my staining in the multiwell dish then fish the section out and mount it on a slide for CLSM.

Good luck

Ian Johnson

Johnson, I.P. (2001). Rapid estimates of neuron number in the confocal microscope combined with in-situ hybridisation and immunocytochemistry. Brain Research Protocols. 8: 113-125.

Aperghis, M., Johnson, I.P., Patel, N., Khadir, A., Cannon, J. & Goldspink, G. (2003). Age and diet influence the survival of injured facial motoneurons Neuroscience 117: 97-104.

Aperghis, M., Johnson, I.P, Cannon, J. Yang, S.Y. & Goldspink, G. (2004). Different levels of neuroprotection by two insulin-like growth factor-1 splice variants. Brain Research 1009: 213-218.

Hi Andre,

We buy the gelatin from SIGMA, and we prepare it as a 10% gelatin-10% sucrose in PBS solution. Also, when I cryoprotect my tissue, I only go up to 10% sucrose, not 30% like you'd do when using OCT. I changed to the gelatin because i found it's much simpler to cut the gelatin than the oct (OCT rolls up too much for my taste).

The brain molds are like these:

http://www.zivicinstruments.com/mouse-brain-slicers-matrix-section.html

They are very useful if you don't want to put the whole brain in the cryostat but have issues cutting it freehand.

If you want the complete gelatin freezing protocol let me know, but you basically need to embed the tissue in the gelatin, using little molds like you'd use with the OCT, and then rapid-freeze at -50 to -60ºC using methylbutane and dry ice in a cold bath.

Our secondary antibodies are all invitrogen goat anti rabbit and goat anti mouse, now that I think about it. I do have NDS for a donkey anti goat secondary antibody. Like Aaron said, those are the two typical serums you'd need to perform most IFs.

Let me know if you have any other question and best of lucks!

-Daniela