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.

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.

Epithelial cells were collected into RPMI-1640 containing 10% (v/v) heat inactivated foetal calf serum. Samples were processed immediately. An aliquot of the cell suspension was diluted 1:2 with 0.4% trypan blue, and applied to a haemocytometer. The total number of cells, and the percentage of viable cells, was determined under a light microscope, within 15 minutes of collection. Each sample of cells obtained by non-bronchoscopic brushing was processed to allow for use in multiple investigative techniques; 1 × 10⁶ cells were used for cell culture or protein extraction, 0.5 × 10⁶ cells to produce cytospin slides for immunocytochemical studies, and RNA was extracted from the remaining 1 × 10⁶ cells.
For culture, cells were washed once in RPMI-1640 media and the cell pellet resuspended in bronchial epithelial basal media (BEBM, Clonetics, CA) supplemented with bovine pituitary extract (50 mM), insulin (5 mM), hydrocortisone (0.5 mM), gentamicin (0.001%, v/v), amphotericin B (0.0005%, v/v), retinoic acid (0.1 μM), transferrin (10 mM), triiodothyronine (6.5 μM), epinephrine (6.5 μM) and human recombinant epidermal growth factor (EGF: 0.5 μM). The cells were then seeded into a culture vessel (25 cm² growth surface area) pre-coated with a mixture of fibronectin, collagen and bovine serum albumin, and maintained at 37°C in a humidified incubator. Twenty-four hours post-isolation, unattached cells were collected. These cells were reseeded into the same culture vessel, with fresh media containing Ultroser G (2% v/v; BioSepra, CA), a serum substitute. The collection and reseeding of viable unattached cells was repeated at both 48 and 72 hours post isolation. Subsequent cultures were fed every second day and were usually passaged every 13–16 days.
Before the remaining cell suspension was used for protein and RNA extraction, and to produce cytospin slides, the macrophages were removed by positive selection: the cell suspension was added to a culture dish that had been previously coated with CD-68 antibody (Dako, Australia). The plate was incubated for 20 minutes (37°C, 5% CO₂) to allow the macrophages to adhere. The suspended epithelial cells were aspirated from the plate, and the macrophages removed using trypsin (0.25%) for subsequent analysis. The macrophage depleted cell suspension was used to produce cytospins, extract protein, and extract RNA.
Cytospin slides were prepared by centrifuging epithelial cells onto a glass slide in a cytocentrifuge (Hettich). Slides were air dried, fixed in 4% paraformaldehyde for 10 minutes, and then stored at -20°C until required. Immunocytochemical staining of the cytospins was used to confirm the purity of the epithelial sample. Antibodies against cytokeratin (a marker for tissue of epithelial origin), α -smooth muscle actin (a marker of myofibroblasts, myoepithelial cells and smooth muscle cells), smooth muscle myosin (a marker of smooth muscle cells), and vimentin (a marker of mesenchymal cells) were used to confirm epithelial cell phenotype.
Immunocytochemical techniques provide only semi-quantitative data about protein expression, and are not sensitive enough to determine levels of protein expression accurately; therefore protein was extracted for analysis with Western blotting. Protein was extracted from the pelleted epithelial cells by lysing the cells in 200 μl of an SDS extraction buffer (20 mM Tris, 1 mM SDS, 1 mM DTT) in the presence of protease inhibitors (Sigma). A commercial assay (Micro BCA Protein Assay, Pierce Biotechnology) was used according to the manufacturers instructions to determine the concentration of total protein in the cell lysate. For each sample, 50 μg of protein was subjected to 12% SDS-PAGE, and immunoblotted with anti-β-actin antibody. Antibody binding was detected with ECL Plus Western Blotting Detection Reagents (Amersham Biosciences).
Total RNA was extracted from epithelial cells using the QIAGEN RNeasy kit (Vic, Australia). RNA quality and quantity was assessed using the Agilent Bioanalyser (Vic, Australia). RNA was prepared from 18 subjects according to a modified version of the protocol of Baugh et al [6 (link)] and hybridised to microarrays. For our first study, gene expression profiles from 9 mild, asymptomatic asthmatics were compared to 9 healthy children for a total of 18 arrays. Expression of genes in cells from asthmatic and healthy children was compared using Affymetrix Human Genome U133 Arrays (HG-U95Av2), which examine the expression of approximately 23,000 genes. Real-time PCR was used to validate the array data for specific genes. Real-time PCR was conducted as previously described [3 (link)].
Data was reported as mean (SE) and analysed by independent samples t-test. Significance was taken as p < 0.05. The proportion of basal cells was analysed using analysis of variance (ANOVA) to compare the difference between the three phenotype groups.