Anti human cd68 clone kp1

Mice were sacrificed at specific time points and tissues for histopathologic analyses were fixed in 4% (wt/vol) paraformaldehyde at 4°C for 24 h. Human and mouse tissues were processed as follows. Two- to three-μm thick formalin-fixed, paraffin-embedded tissue sections were deparaffinized and rehydrated and subsequently stained with either hematoxylin and eosin stain or a modified Ziehl-Neelsen stain for bacterial detection or immunohistochemistry stain, respectively (24 (link)). For immunohistochemistry, tissue sections underwent a steam pressure antigen retrieval step using Dako target retrieval solution, pH9. Slides were incubated with the following mouse primary antibodies: anti-human CD68 (clone KP1, Dako), anti-human CD15 (BD), anti-human CD4 (clone 4B12, Leica Biosystems), anti-human CD8 (clone 4B11, Leica Biosystems), and anti-human CD20 (clone L26, Leica Biosystems) for 1 h at room temperature in a humidified chamber. The tissue sections were developed with a biotin-free HRP- polymer system (MACH 4 Universal HRP-polymer kit, Biocare Medical) and DAB substrate (Dako). Tissue sections were imaged using a Leica DRMB microscope with a ProgResC12 (Jenoptik) Camera. Lung tissue sections were scanned using Aperio AT2 Leica slide scanner. Further analysis was carried out using ImageJ version 1.41.

Paraffin sections of FPPE samples were processed in a fully automated staining instrument ULTRA (Ventana Medical Systems) using the Ultra-view kit. The following antibodies were used: Anti-human CD68 clone KP1 from DAKO (1/1,000) detects a monocyte/macrophage associated antigen. Anti-human CD34 clone QBEnd/10 from Novocastra (1/40) predominantly stains endothelial cell membranes. Anti-human CD163 clone 10D6 from DBS (1/75) is specific to the monocytic-macrophage lineage. Anti-human Mast Cell Tryptase (MCT) clone AA from DAKO (1/1,600) is specific for mast cells. Slides were scored by consensus of four simultaneous experienced readers and graded from −2 to +4 by comparison with adipose tissues from a normal breast reduction reference. Means and standard deviations of the four scores in samples AdipN, AdipTa, and AdipTd were computed. A paired Wilcoxon test was performed to test for differences between AdipTa and AdipTd and a Wilcoxon test to test for difference between AdipN and AdipTa. Spearman's correlations ρ between scores in samples AdipTa and AdipTd were computed, and their significance was tested using the cor.test R function [R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/].