Immpact novared kit

Formalin‐Fixed Paraffin‐Embedded (FFPE) patient brain tissue sections were first deparaffinized and rehydrated. Sections were quenched with 1% H₂O₂ followed by antigen retrieval. Permeabilization was done using 0.1% Triton‐X in 1X PBS and later on blocked with 1% BSA, 3% goat or rabbit serum, 0.05% Triton‐X in 1X PBS. Sections were incubated overnight with primary antibodies, goat anti‐coronin 1A (Abcam, 1:25, Cat# ab14787, RRID:AB_301480), rabbit anti‐GFAP (Dako, 1:500, Cat# Z0334, RRID:AB_10013382) in blocking solution. Sections were washed 4 times with 0.02% Tween 20 in 1X PBS (PBST). Secondary antibody incubation was done for 2 h with appropriate biotinylated antibody (Vector labs, 1:500). Washing was done 4 times using PBST, and sections were then treated with a complex of Avidin‐Biotinylated HRP (Vector Labs, 1:1, Cat# PK‐6100) for 2 h followed by 4 PBST washes. Sections were developed for 1–3 min using the ImmPACT NovaRED kit (Vector Labs, Cat# SK4805) following the manufacturer's protocol, counterstained with hematoxylin, and washed under running water. The sections were dried, treated with xylene, and then mounted using the DPX mountant solution. Each section was imaged thrice randomly using a Leica DMRXA2 (Leica) microscope at 10x and 40x magnifications. Images were then processed and analyzed using the ImageJ software (NIH, RRID:SCR_003070).