Gelatin Embedding of Fixed Brain Tissue
(adapted from Trygve Leergaard, University of Oslo)
Pre-sink brain in 30% sucrose prior to embedding
Remove meninges. Rinse brain in PBS. Submerge brain in 5% gelatin (gelatin from porcine skin, 300 bloom, Sigma, G2500) in PBS and incubate at 37° for 2 hours.
Move brain to 9% gelatin in PBS and incubate at 37° for 2 hours.
While brain is incubating in gelatin, pour a gel platform (using 11-13% gel) about 2mm thick in the bottom of the mold. Allow to harden at room temperature.
Position brain on gel platform. Fill mold with fresh 11-13% gelatin until it covers the brain by 2-3mm, being careful to avoid bubbles in the gel. Allow to harden at room temperature.
**Pour slowly! Pouring the gel too fast will cause your brain to shift positions.
Remove gelatin from gel and fix in 4% PFA overnight at 4°.
NOTE: for YFP/GFP fix for only 4 hrs.
Sink gelatin block in 10% sucrose overnight at 4°.
Submerge gelatin block in 30% sucrose at 4°until it sinks (2-3 days, depending on size of gel block).
Sectioning
Use large stage for microtome (Leica #14050842641 dry ice tray) to ensure adequate temperature can be maintained.
Pour about 1” isopentane into a beaker and cool to -60° on dry ice—you can put a few chunks of dry is in the isopentane to help cool it if you need to.
Trim gel block to leave ~5mm gel on each side of brain. Place in large peel-a-way mold (Polysciences Inc., 18648A-1) and surround with 2% gelatin in PBS (this creates a nice gel/PBS interface around the tissue block to make sectioning easier).
Slowly lower the mold into the isopentane, making sure the isopentane doesn’t spill over into the mold. Leave in isopentane until the block freezes through (5-10 min).
Peel away the plastic mold and mount frozen block on microtome using PBS.
Alternatively: Bob Switzer says using 10% alcohol in water on the stage instead of PBS will help make sure your gelatin block is mounted straight. The alcohol/water mix will spread out more than PBS, allowing you to avoid a PBS mound that might elevate the block unevenly. Store sections in antifreeze at -20°
Mounting / Staining
Use NSA solution to mount (ammonium acetate buffer, see below for recipe). If the gelatin seems like its too big for the brains, the pH is too low which has caused your tissue to contract. Waft over ammonium hydroxide and then switch to a higher pH solution.
The sections generally perform well for free-floating protocols, EXCEPT when an acid pretreatment is required (i.e., for Perl’s stain or formic acid for amyloid). Acidic treatments can make the sections to stick together in a big ugly clump. Conversely, Thioflavine treatments can make the tissue crinkle within the gelatin and become hard to mount flat. For these protocols, mount sections onto a slide BEFORE staining, let dry overnight at RT, then rehydrate the following day and ensure the tissue is really adhered to the slides by bringing up to 95% ethanol, then immersing for 5 min in 95% ethanol / formalin(9:1 EtOH:37% formalin), then rinsing in 95% ethanol and hydrating down to water prior to staining.
The gel can cause unusually high background with immunostains, so you may need to add a second blocking step prior to incubation in secondary antibody.
NeuroScience Associates Mounting Solution
Ammonium acetate stock solution:
1 M acetic acid: 15 ml glacial acetic in 235 ml H2O (glacial acetic is 17.4 M)
1M ammonium acetate: 19.27 g ammonium acetate in 250 ml H2O
pH 6.0(50 ml final volume): 1.875 ml 1M acetic acid
48.1 ml 1 M ammonium acetate
Start with pH 6 for most sections.
pH 4.9(50 ml final volume): 15 ml 1M acetic acid
35 ml 1 M ammonium acetate
Low pH solution not used very often.
Subbing solution: 0.5% gelatin in dH2O, dissolved at 60º C
Ammonium acetate WORKING solution for mounting gelatin sections:
12 ml Ammonium acetate stock buffer (pH 4.9 or 6.0)
50 ml 95% ethanol
2 ml Subbing solution
188 ml H2O