TITLE: Immunofluorescence for free-floating brain sections
AUTHORS: Jill R. Crittenden, Tomoko Yoshida, Margaret I. Davis, Ann M. Graybiel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to prepare fixed brain sections and process them for immunofluorescence.</div></div>

[STEPS]
?. [Brain tissue preparation]
Brains are dissected from deeply-anesthetized mice that were trans-cardially perfused with 10 ml of room-temperature 0.9% saline followed by 60 ml of 4% PFA. PFA must be used with appropriate protection and ventilation. Brains are then post-fixed in 4% PFA in glass vials for 2 hours with gentle rocking at 4 °C. Replace PFA solution with glycerol cryoprotectant solution until the brain sinks (overnight), at 4 °C. Store the brains in the same solution at 4 °C until sectioning. For sectioning, remove the brains from storage cryoprotectant solution, freeze in powdered dry ice on a chuck, and cut at 30 μm intervals on a sliding microtome. Put sequential sections into adjacent wells of a multi-well plastic tray containing 0.1% sodium azide in 0.1 M PB for storage at 4 °C until immunostaining.
Paraformaldehyde (PFA) is toxic, a strong irritant, and flammable as a solid. Prepare and use solutions only under adequate ventilation and with eye and skin protection.https://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=158127&brand=SIAL&PageToGoToURL=https%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2F158127%3Flang%3Den
?. [ Immunofluorescence reactions ]
Rinse sections in PBST, 3 x 2 minutes
?. Incubate in blocking solution, 20 minutes
?. Incubate in primary antibodies diluted in blocking solution, 1 overnight at 4oC
?. Rinse in PBST, 3 x 2 min
?. Incubate in secondary antibodies diluted in blocking solution, 2 hours at room temp. in dark[We use Alexa fluorophore conjugated secondary antibodies (Thermo Fisher Scientific), diluted 1:300]
?. Rinse in 0.1M PB, 3 x 2 minutes
?. Submerge sections in mounting solution in a large glass Petri dish and use a paintbrush to move them onto glass microslides (VWR #48311-703)
?. Let sections sit flat, covered loosely by foil, until they appear almost dry, 15 - 60 minutes, depending on the room humidity
?. Coverslip with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific cat. #P36931)
?. [Solution preparation]
0.4 M NaKPO4 Phosphate Buffer Stock (dilute this to make 0.1M PB for PFA, mounting solution, and rinse buffer)112 g   K2HPO4, (potassium phosphate dibasic; MilliporeSigma cat. # PX1570)21.2 g Na2H2PO4H2O (sodium phosphate monobasic; VWR cat. #BDH9298)2 L       Distilled H2O0.1 M NaPO4 Phosphate Buffer Stock (use this to make PBST)23.1 g  Na2HPO4 (sodium phosphate dibasic;VWR cat. # 0404)5.04 g NaH2PO4H2O (sodium phosphate monobasic;VWR cat. # BDH9298)PBST working solution200 ml             0.1M NaPO4 Phosphate Buffer Stock for PBST1800 ml           Distilled H2O17.5 g              NaCl sodium chloride, Granular (Macron Fine Chemicals cat. #7532)0.4 g                KCl potassium chloride (Macron Fine Chemicals cat. #6858)4 ml                 Triton X-100 (Sigma cat. # T9284)4% PFA (freshly depolymerized from paraformaldehyde) for perfusion Prepare in a fume hood, formaldehyde is a biohazard.Add 4 % w/v paraformaldehyde (Macron Chemicals cat. #2621-59) to 0.1M PB.Heat mixture to 80-85ºC (do not let it go beyond this temperature).Remove from heat and stir with a stir-bar until all solids have dissolved and the solution is clear.Allow to cool to room temperature, then filter the solution through filter paper (VWR cat. #28331-081).If using the same day, keep at room temperature, otherwise, store at 4 ºC.Glycerol cyroprotectant solution25 ml   glycerol (VWR cat. #0854)75 ml   0.1M PB with 0.1% sodium azide (MP Biomedicals cat. #0210289190)TSA blocking bufferHeat 500ml PBST to 50°CAdd 2.5g Blocking Reagent (0.5% final; Perkin Elmer cat. #FP1012)Stir until dissolved and cool to room temperature.Use a funnel to filter through paper (VWR cat. #28331-081).Mounting SolutionHeat 500ml 0.1M PB with 0.1% sodium azide to 55°CAdd 0.4g gelatin (0.08% final;Sigma cat. #G1890)Cool to room temperature.Use a funnel to filter through paper (VWR cat. #28331-081).
Paraformaldehyde (PFA) is toxic, a strong irritant, and flammable as a solid. Prepare and use solutions only under adequate ventilation and with eye and skin protection.https://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=158127&brand=SIAL&PageToGoToURL=https%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2F158127%3Flang%3Den
?. Store slides in the dark at 4 0C