Briefly, the mouse brain tissue sections are prepared by washing off the cryoprotectant medium and then antigen retrieval is performed followed by quenching, blocking and primary antibody incubation. Sections are then washed and incubated in the appropriate secondary antibody solution and are then mounted, cover-slipped and sealed.

30 μm mouse brain sections were stored in anti-freeze solution at -20°C until required.

1. Pour sections into a well insert in a 6-well plate to separate storage solution from section
2. Move the well insert to another well containing approximately 6mL of 1x PBS. Wash at least 6x with 1x PBS for 0h 5m 0s each on an orbital shaker using low speed at

Place sections in labelled glass vials containing 6mL 1x citric buffer (CB) pH6.0.1. Place in the steamer (Breville, Model: BFS800BSS) on high for 0h 22m 0s 98°C.

1. Cool down to Room temperature
2. Place the sections back into its corresponding wells.
3. 1xPBS wash: 2 x 0h 5m 0s

Incubate in blocking buffer: 2h 0m 0s Room temperature on shaker60rpm2. Make primary antibody cocktail in home-made IF buffer

3. Label ceramic plate and place sections in antibody cocktail: 72h 0m 0s at 4°C on the shaker

• AT8(Ms IgG1), Thermofisher #MN1020 1:200

• SP70 (rb), Sigma #SAB5500182 1:200

• TH (Ms IgG2b), Thermofisher # TA506549 1:200

Transfer sections from ceramic plate to well plates1. 0.1% PBST wash: 3 x0h 10m 0s

1. Make secondary cocktails as below in IF blocking buffer

• Gt @ Ms IgG, ThermoFisher #A-21206 1:250

• Dn@Rb-AF568, ThermoFisher #A-10042 1:250

• Gt@Ms IgG2b-AF647, Thermofisher #A-#A-21242 1:200

• Hoechst 33,342 (1mg/ml stock) Sigma #B2261 1:1000

4. Place sections and antibody cocktails in a ceramic plate: 2h 0m 0s Room temperature on the shaker 60rpm

5. Place sections back into wells

6. 0.1% PBST wash: 3 x 0h 10m 0s

7. 1x PBS wash: 3 x 0h 5m 0s

Label slides with corresponding section and antibody information1. Place sections in a petri dish with 1xPBS and nudge the sections onto slides with brush

1. Mount with anti-fade media (DAKO Fluorescence Mounting Medium, Agilent, cat# S302380-2)
2. Wait for sections to become dry and seal with sealant (Biotium CoverGripTM Coverslip Sealant, cat#23005)
3. Store in slide box and store in fridge/cool room 0h 5m 0s