Cd68 antibody

COVID 19 patient autopsy lung samples were procured from the UCLA Translational Pathology Core Lab for research use. Samples were processed and sectioned, after a pathologist confirmed the section quality by H&E staining, and subsequent confirmation of COVID-19 positivity by RNAscope V-nCoV2019-S probe (ACD, Cat#: 848568, ready to use). Immunohistochemistry stainings were also performed on this lung tissue: Paraffin-embedded sections were cut at 4μm thickness and paraffin was removed with xylene and the sections were rehydrated through graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. Heat-induced antigen retrieval (HIER) was carried out for all sections in AR9 buffer (AR9001KT Akoya) using a Biocare decloaker at 95°C for 25 min. The slides were then stained with YAP (S127) antibody (Cell Signaling, 13008, 1–100) and CD68 antibody (Dako, m0876, 1–200) at 4 degree overnight, the signal was detected using Bond Polymer Refine Detection Kit (Leica Microsystems, catalogue #DS9800) with a diaminobenzidine reaction to detect antibody labeling and hematoxylin counterstaining.

Subcutaneous AT samples from 45 lean and 47 overweight and obese children included in the previously described Leipzig Childhood Adipose Tissue cohort [12 (link)] were obtained during elective surgery. Children underwent detailed anthropometric, clinical and metabolic assessments [12 (link)]. The study was approved by the ethics committee of the Medical Faculty, University of Leipzig (Reg.No: 265-08-ff; NCT02208141) and written informed consent was obtained from all parents.
Preparation of and analyses of AT samples was performed according to previously published protocols [12 (link)]. Briefly, adipocytes and stromal vascular fraction (SVF) were isolated by collagenase digestion and adipocyte diameter was determined after osmium fixation using a Coulter counter (Multisizer III; Beckmann Coulter). Macrophage infiltration was analysed by immunohistochemical staining of AT sections with CD68 antibody (M0718, DAKO).
Prior to surgery, fasting blood samples were obtained and stored at -80°C. Analyses of serum parameters (adiponectin, leptin, high sensitivity C-reactive protein (hsCRP), glucose and insulin) were performed by a certified laboratory (Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig).

COVID-19 patient autopsy lung samples and normal lung samples (n = 4 each) were procured from the UCLA Translational Pathology Core Lab for research use. Samples were processed and sectioned, after a pathologist confirmed the section quality by H&E staining, and subsequent confirmation of COVID-19 positivity by RNAscope V-nCoV2019-S probe (ACD, Cat#: 848568, ready to use). Immunohistochemistry stainings were also performed on this lung tissue: Paraffin-embedded sections were cut at 4-μm thickness and paraffin was removed with xylene and the sections were rehydrated through graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 minutes. Heat-induced antigen retrieval [9 (link)] was carried out for all sections in AR9 buffer (AR9001KT Akoya) using a Biocare decloaker at 95°C for 25 minutes. The slides were then stained with YAP (S127) antibody (Cell Signaling, 13008, 1–100) and CD68 antibody (Dako, m0876, 1–200) at 4 degree overnight; the signal was detected using Bond Polymer Refine Detection Kit (Leica Microsystems, catalogue #DS9800) with a diaminobenzidine reaction to detect antibody labeling and hematoxylin counterstaining.