*4.5. Immunohistochemistry Staining*

Paraffin embedded tissue sections, collected as described in the RNA-scope methods section, were incubated at 60 ◦C for 20 min. Immediately after incubation, the sections were deparaffinized with three washes in xylene for 5 min each, followed by three washes in 100% ethanol for 3 min each, one wash in 95% ethanol for 1 min and rinsing the slide under running deionized water (DI) for 3 min. Antigen retrieval was achieved by placing the slides in sodium citrate buffer (pH 6) for 40 min at

100 ◦C in a water bath. The slides, along with sodium citrate buffer were allowed to cool down to room temperature (RT) for 30 min and washed two times in PBST buffer (0.1% tween20 in PBS pH 7.4) for 5 min each wash. The sections were blocked with hydrogen peroxide (Fisher scientific NC0185217) for 10 min and washed two times with PBST buffer, 5 min each. The sections were incubated at RT for 1 h in blocking buffer (1% DMSO and 1% BSA in PBS buffer). After draining the blocking buffer, primary staining was performed using the CD34 antibody (Abcam ab81289, Cambridge, UK) at a 1:400 dilution in blocking buffer, and slides with primary antibody were incubated overnight at 4 ◦C in a humidified chamber. Following primary antibody staining, the sections were washed in six changes of PBST for 5 min each and incubated for 1 h at RT in secondary Goat anti-rabbit HRP antibody (Abcam ab6112) at a 1:750 dilution in blocking buffer. This was followed with three washes in PBST, for 5 min each. The staining was visualized using DAB for 10 min (Fisher scientific NC9276270, Hampton, NH, USA) following the vendors recommended protocol and washing away excess DAB under running DI water for 5 min. The sections were counter stained with Mayer's Hematoxylin (Fisher scientific 5031794) for 7 min at RT and incubated in bluing agent (Fisher scientific 22050114) for 2 min. The sections were washed in DI water and patted dry before mounting.
