Another alternative for long term storage of 4%PFA fixed rat brain sections (we have 30uM thick coronal sections) is to place them in a cryoprotectant solution (immersed) then store them at -20Celcius in a frost free freezer. The recipe is as follows:

30%Ethylene glycol, 20% glycerol, in a 0.05M NaPhosphate buffer pH7.4. We have kept 6 sets of serial sections in this solution for 3 years and still had robust immunohistochemical detection of proteins.

Definitely too long. You can replace PFA with PBS/sodium azide. With that you can store them for many many months before cutting them at a vibratome. Here's the recipe

Sodium Azide: (highly toxic – wear two pairs of gloves and lab coat – careful not to breath in)

0.025% in PBS (used to store sections and for immunos)

Thank you Mohamed. I read the discussion  as you suggested me. I found it interesting that although my brains have a long post-fixation (which, according to the discussion, it  cause cross-linking of the protein at lysine residues), when I made IHC against a histone, acetylated in a specific lysine,  I found staining. 

I used antigen retrieval protocol and the antibody  bound to the lysine.

I know little on the subject of post-fixation, but perhaps as suggested here, the effect of post-fixation depends on the tissue and the antibody. 

Thanks Giulio. Can this material be stored at 4 ° C? and another question, I cut the brains in a cryostat, would it be appropriate to use Sodium Azide and then cut into cryostat?

Hi, sorry I did not understand you had to cut with a cryostate. Yes, you can store at 4C the brains in PBS/Azide as I said. Alternatively, assuming you had cryoprotected your brain with sucrose, one thing you can do afterward is to embed the brains in oct and store the entire block in -20C until you cut it. I used to do that with embryonic brains.

Thank you.

Another alternative for long term storage of 4%PFA fixed rat brain sections (we have 30uM thick coronal sections) is to place them in a cryoprotectant solution (immersed) then store them at -20Celcius in a frost free freezer. The recipe is as follows:

30%Ethylene glycol, 20% glycerol, in a 0.05M NaPhosphate buffer pH7.4. We have kept 6 sets of serial sections in this solution for 3 years and still had robust immunohistochemical detection of proteins.

We had a rat brain which was stored for 6 months in 4% PFA. The brain was stored in didactic aim to show cryostat sectioning and immunohistochemistry to students. I was very surprised when immunohistochemical (IHC) procedure of c-Fos protein detecting was successful after too long storage in PFA. But normally we store brains for 24h in PFA in room temperature for c-Fos detecting IHC procedure. For other IHC and immunofluorescent detections even 1h is appropriate time.

Another possibility: 30% sucrose solution with a drop of Azid.

It's too long. I think the best for a long term storage is that the brain is fixed in 4% PFA, then post-fixed for 48 h to overnight max at 4 °C and finally stored at -18 °C in a cryoprotectant solution for later use. Furthermore, cryoprotection is important if you use a cryostat in order to avoid formation of ice crystal in your sample when you cut it.

In fact, the time of post-fixation depends on the type of elements you want to detect.

Thank you every one. I will put into practice the brains' immersion in cryoprotectant solution.

HI,

The best way to store brain is to immerse the brain in OCT overnight or 24 hour in a shaker and freeze the brain on dry ice and store it in -80 °C. If brain is store in 30% sucrose/PBS for long time, the brain is dehydrated and shrank, we tested this using MRI a couple of years ago.

Hi Ginna,

I think the key is how long will you storage those rat brains? Up to 4 months I would use 30% sucrose in PBS 0.1M. More than 4 months I would use cryoprotectant solution and put them in -20 C. However, keep in mind the suggestions from Xiaoying because dehydration could affect what you are looking for, it depends of your experimental question. I would not keep those brains in only 4% PFA more than a week, this could affect the expression of your molecule, although it depends of its molecular features. Good luck!

Personally, I think that the best is to keep them at 4C in 20% sucrose solution with or without sodium azide. In that way, they will stay fresh for many months/years even. My best staining results were obtained from tissues stored like that. The general procedure is: post-fix in 4% PFA for max 24h, rinse 3x in PBS, transfer to sucrose solution and store in the fridge (up to 5 years) for further procedures. Freeze in OCT only if you plan to cut the tissue shortly after (within 1-2 weeks). OCT molds tends to shrink over years (even if kept in - 80C) and after 2-3 years the tissue is useless while in sucrose it can sustain for much longer time. Off note - if you decide not to use sodium azide then you need to change sucrose solution every 1-2 months to prevent bacterial/fungal growth.

I agree with Judyta. Storing rat brains for a long time can shrink the tissue. Couple of months are OK. So I store them in 30% Sucrose solution and keep them at 4 C. If needed I add Sodium Azide to protect them from growing molds. But I prefer to section them as soon as possible and store them at 4C  in PBS solution (12 or 24 tissue culture well plates serially numbered) containing 2 mM EDTA. They can be stored this way for at least a month if not more. Hope it helps.

Mouse brains: I post fix for overnight at 4 deg. then rinse and store in buffer with an 1/2 ml of fix per 50ml of buffer no longer than 2-4 weeks before processing.  The fix is just to keep mold down.  But I have even seen mold grown in that when I kept the remains from an experiment "just in case" at a request from a student.  They went on and I forgot the jar in the refrigerator for about 3-4 months and it had little green things.  Surprise!

I usually process within the 2 week time frame.  Sucrose would be ideal.  A nice tip: start with 10% for overnight then, 20 then 30.  Seems to allow penetration into the ventricles and less distortion of the cells. 

As said in the responses, long postfixation is not good for IHC. Let it in sucrose at 4°C is a good alternative. I prefer to freeze the sample and to store it at -80°C (more than 1-2 years) or if not possible at -2O°C (for many months). 

All the suggestions given above will help you certainly to preserve your sample. If you have not the possibility to freeze your sample, you can keep it in PBS with sodium azide at 4°C for several months or PBS alone at 4°C but with renewing the solution of PBS 1time/week or 10 days.

For IHC the shorter the better for most of the antigens. I have best results with 3h in PFA 4% at 4ºC followed by over-night in Sucrose 15% PBS. Finally cut in sliding microtome (frozen).

If you are afraid of loosing antigenicity the time before you can cut your "long stored" samples add one or two fresh brains with short post fix and compare. In worst scenario compare your 1 year post-fix with the "months" brains.

In my hands acetilated histone IHC is tricky and never had good results.

Good luck.

Can a mouse brain be stored in PFA for longer than 24 hours but shorter than a week? I plan to put them in PFA on a Friday and do sucrose the followingredients Monday.

Hey Jessica. I've left brains in PFA for 2 days and the result has been the same. I don't believe you need to to worry about 3-4 days. If you are fixing the brain for histological purposes, I suggest antigen retrieval to ensure that the prolonged fixation time doesn't skew your results.

I have used  rat brain stored in 4% PFA  for more than couple of months with periodically changing the PFA. The brain sections were used for  Nissl stain and H&E, found no issues in staining i.e stained sections and histological images are good.  

I have used 4%PFA (for mouse tissues and zebrafish 5dpf embryos) to fix the tissues at 4 °C for few weeks, and also, fixing overnight followed by dehydration in ethanol for several weeks.

I did not see any significant changes to the tissues.

an someone suggest the best protocol for antigen retrieval in brain of rat post-fixed for histology?