Immunohistochemistry

Immunohistochemistry 

Brains from indicated littermates were fixed using 4% paraformaldehyde (PFA) at 4°C overnight with gentle agitation, cryopreserved in 30% sucrose, frozen, and finally stored at −20°C until use. For staining, 20-mm cryosections were made and incubated in blocking/ permeabilization solution containing 3% normal goat serum (NGS) and 0.2% Triton-X in PBS. The sections were treated overnight with appropriate primary antibodies diluted in 1% NGS/0.2% Triton X-100/PBS followed by the corresponding secondary antibodies for 2 hours at room temperature. The following primary antibodies were used: rabbit anti-CD68 (Abcam); mouse anti-MHC-II (Abcam); rabbit anti-Ki67 (Abcam); isolectin GS-IB4, Alexa Fluor 594 conjugate (Invitrogen); rabbit anti-TNFa (Abcam); rabbit anti-Iba1 (Wako Chemicals USA Inc.); rabbit anti-Tmem119 (Abcam); and rat antiP2RY12 (BioLegend).

Detail protocol:

1. Brains from indicated littermates were fixed using 4% paraformaldehyde (PFA) at 4°C overnight with gentle agitation, cryopreserved in 30% sucrose, frozen, and finally stored at −20°C until use. 
2. 1XPBS, 5 min wash, each time, x3
3. 0.2% Triton X-100, 15 min
4. 3% NGS in 1XPBS, 60 min
5. Primary Ab (1:500 dilute; rabbit anti-Tmem119 (Abcam, ab209064)), 4°C, overnight (at least 16 hrs)
   *Primary antibody solution:
   1%NGS
   0.2% Triton-100
    Primary antibody
6. 1XPBS, 5 min wash, each time, x3
7. Secondary antibody (1:500 dilute), RT, 3hrs
8. 1XPBS, 5 min, x3
9. Mounting solution with DAPI

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