Horseradish peroxidase hrp conjugated goat anti rabbit antibody

The antibodies (and their Env epitopes) used in this study include the bNAbs VRC01, VRC03, 3BNC117 and b12 (CD4-binding site, CD4BS); PGT145 and PG9 (V2 quaternary, V2q); PGT151 and 35O22 (gp120–gp41 interface); 2G12 (gp120 outer domain glycans); and 10E8 (gp41 MPER). The pNAbs used in this study include 19b and 39F (gp120 V3); b6 and F105 (gp120 CD4BS); 902090 (gp120 V2 linear); 17b and E51 (gp120 CD4-induced, CD4i); and F240 (gp41 Cluster I). CD4-Ig is a fusion protein consisting of the N-terminal two domains of human CD4 and the Fc portion of an antibody^{14 (link)}. Antibodies against HIV-1 Env were kindly supplied by Dennis Burton (Scripps), Peter Kwong and John Mascola (Vaccine Research Center, NIH), Barton Haynes (Duke University), Michel Nussenzweig (Rockefeller University), Hermann Katinger (Polymun), James Robinson (Tulane University) and Marshall Posner (Mount Sinai Medical Center). In some cases, anti-Env antibodies were obtained through the NIH AIDS Reagent Program. Antibodies for Western blotting included goat anti-gp120 polyclonal antibody (Thermo Fisher) and the 4E10 anti-gp41 antibody (Polymun). A horseradish peroxidase (HRP)-conjugated rabbit anti-goat antibody (Thermo Fisher) and an HRP-conjugated goat anti-human IgG antibody (Santa Cruz) were used as secondary antibodies for Western blotting.

Immunoprecipitation assays were performed using a Classic IP Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. NSC34 cells were plated at 1.4 × 10⁵cells/well in 6-well plates (BD Biosciences) and incubated overnight. The cell lysates were incubated with mouse anti-NKA α1 antibody, mouse anti-NKA α3 antibody, or normal mouse IgG (Santa Cruz Biotechnology). A mixture of equal parts of a protein sample and sample buffer with 20% 2-mercaptoethanol (Wako, Osaka, Japan) was subjected to SDS-PAGE (Wako). The separated proteins were then transferred onto a polyvinylidene difluoride membrane (PVDF, Immobilon-P; Merck KGaA, Darmstadt, Germany). To visualize the proteins on the membrane, the primary antibody was goat anti-GPNMB antibody (1: 500, R&D Systems Inc., Minneapolis, MN, USA) and the secondary antibody was horseradish peroxidase (HRP)-conjugated rabbit anti-goat antibody (1: 2000, Thermo Fisher Scientific). The immunoreactive bands were visualized using a chemiluminescent substrate (ImmunoStar^® LD; Wako). The band intensity was measured using an imaging analyzer (LAS-4000; Fuji Film, Tokyo, Japan).

Lungs were fixed in 4% PFA for 2 hours and placed in a 30% PBS sucrose solution overnight at 4 °C before being embedded in a cutting medium and frozen at −80 °C. Serial 8-µm transversal cryo-sections of lungs were obtained. VCAM-1 expression was analyzed using rabbit polyclonal anti-VCAM-1 antibody (sc-8304) from Santa Cruz Biotechnology. For detection, horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody was used (Thermo Fisher Scientific). Sections stained with secondary antibodies alone were used as controls of non-specific staining. The slides were examined under a light microscope. Lung sections were further counterstained with hematoxylin and eosin and examined at 20× or 63× magnification using a DMi8 advanced fluorescence microscope (Leica Microsystems, Germany). NIH ImageJ software version 1.45 was used for quantification.