Background reducing antibody diluent

Staining for SARS-CoV-2 antigen was achieved on the Bond RX automated system with the Polymer Define Detection System (Leica) used per manufacturer’s protocol. Tissue sections were dewaxed with Bond Dewaxing Solution (Leica) at 72 °C for 30 min then subsequently rehydrated with graded alcohol washes and 1× Immuno Wash (StatLab). Heat-induced epitope retrieval (HIER) was performed using Epitope Retrieval Solution 1 (Leica), heated to 100 °C for 20 min. A peroxide block (Leica) was applied for 5 min to quench endogenous peroxidase activity prior to applying the SARS-CoV-2 antibody (1:2000, GeneTex, GTX135357). Antibodies were diluted in Background Reducing Antibody Diluent (Agilent). The tissue was subsequently incubated with an anti-rabbit HRP polymer (Leica) and colorized with 3,3’-Diaminobenzidine (DAB) chromogen for 10 min. Slides were counterstained with hematoxylin.

Tissue sections were deparaffinized and rehydrated using Tissue-Tek Tissue Clear and decreasing concentrations of ethanol, followed by rinsing in water. Sections for IHC were pretreated with 5 µg/ml proteinase K (Roche) in 50 mM Tris, 0.5 M EDTA, pH 8 at 37 °C for 15 min, followed by blocking of endogenous peroxidase activity with 1% H₂O₂ for 15 min. Immunostainings were performed using rabbit anti-RFP pAb (2 µg/ml, ROCKLAND, # 600–401-379) and rabbit anti-MT1-MMP mAb (3.7 µg/ml, Abcam, ab51074, EP1264Y). Primary antibodies were diluted in Background Reducing Antibody Diluent (Agilent). Sections were incubated with primary antibodies overnight at 4 °C, washed in TBS-T (50 mM Tris, 150 mM NaCl, 0.5% Triton X-100, pH 7.6) and incubated 45 min with EnVision + System HRP Labelled Polymer Anti-Rabbit IgG (Agilent). Chromogen staining was performed using NovaRED HRP substrate kit (VWR). Nuclear counterstaining was done using Mayer’s haematoxylin (Histolab). After staining, sections were dehydrated with increasing concentrations of ethanol, followed by Tissue-Tek Tissue Clear and mounted using Tissue-Tek Tissue Mount (Sakura). TRAP staining was performed as described^{14 (link), 48 (link)}. Overview pictures were obtained with a Nanozoomer Digital Pathology slide scanner and close-up pictures were taken with a camera connected to a Leica DM2500 microscope.

Five micron formalin-fixed, paraffin embedded sections were cut for immunostaining and analyzed as previously described [63 (link)]. IHC staining was performed at the Pathology Research Core (Mayo Clinic, Rochester, MN) using the Leica Bond RX stainer (Leica). Briefly, tissue slides were dewaxed and retrieved on-line using the following reagents Bond Dewax (Leica) and Epitope Retrieval 2, EDTA based (Leica Biosystems Inc. Buffalo Grove, IL). Tissue slides were retrieved for 30 minutes. The ERβ1 PPG5/10 antibody (Thermo Scientific, Waltham, MA) was diluted 1:75 in Background Reducing Antibody Diluent (Dako, Agilent Technologies, Santa Clara, CA) and incubated for 30 minutes. This antibody has been shown to be highly specific and sensitive for detection of only the full-length form of this receptor in previous IHC studies [59 (link)-61 (link)].

All chromogenic immunohistochemistry (IHC), immunofluorescence (IF), and in situ hybridization staining was performed on the Leica Bond RX automated system. All tissue sections were dewaxed for 30 min in Bond Dewax Solution (Leica Microsystems) heated to 72 °C. Tissues were then rehydrated with absolute ethanol washes and 1x ImmunoWash (StatLab). All antibodies were diluted in Background Reducing Antibody Diluent (S3022, Agilent).