Rat anti ly6b 2

Immunohistochemical procedures were performed as previously described^{54 (link)}. Sections were probed for 40 h at room temperature with rat anti-CD4 (1:500, BD Bioscience, San Jose, CA, USA), rat anti-CD68 or rat anti-Ly6B.2 (1:1,000, AbD Serotec, Bio-Rad, Raleigh, NC, USA), goat anti-5-HT (1:5000, Immunostar, Hudson, WI, USA), rat anti-CD117 (1:500, R&D Systems, Minneapolis, MN, USA), or sheep anti-calcitonin gene-related peptide (CGRP) antibody (1:4000, Enzo Life Sciences, Farmingdale, NY, USA). After washing in PBS, sections were incubated for 4 h at room temperature with corresponding secondary antibody. Quantitative determinations were made from three random locations for each mouse. For analysis, the number of CD4-, CD68-, Ly6B.2-, 5-HT-, and CD117-immunopositive cells, and CGRP-immunopositive neurons were counted per 10⁶ µm² area of tissue.

The primary antibodies used were: rabbit anti-XB130 (1:1,000, Abgent, San Diego, CA), rabbit anti-Ki67 (1:100, Lab Vision, Fremont, CA), rat anti-Ly-6B.2 (1:10,000, AbD Serotec, Raleigh, GC), rat anti-F4/80 (1:500, AbD Serotec), goat anti-podoplanin (PDPN) (1:50, R & D Systems, Minneapolis, MN), rabbit anti-surfactant protein C (SFTPC, for type II cells)(1:1,000, Seven Hills Bioreagents, Cincinnati, OH) and rabbit anti-phospho-GSK-3β-Ser9 (1:500, Cell Signaling, Beverly, MA). After incubation with primary antibodies, sections were incubated with appropriate secondary antibodies. IHC was performed using a Vectastain ABC kit (Vector Laboratories, Burlington, Canada) with 3-3-diaminobenzidine as chromogen, and sections were counterstained with hematoxylin, and images were captured via Olympus BX51-FL. For IF, the secondary antibodies used were: donkey anti-goat Alexa Fluor^® 488, donkey anti-rabbit Alexa Fluor^® 555 and goat anti-mouse Alexa Fluor^® 555 (1:200, Invitrogen, Burlington, Canada), and sections were mounted with Prolong Gold Antifade Mountant with DAPI^® (Invitrogen). The slides were examined with an Olympus BX-51, and images were captured via QImaging colour camera (Olympus Co, Ltd). We randomly chose 5 fields (×200) per slide for positive cell counting.