Anti mouse horseradish peroxidase hrp conjugated secondary antibody

The plasma protein (20 μg) from CAD and healthy controls was separated on 12% SDS-PAGE as described previously [25 (link)] and electrotransferred to the nitrocellulose (NC) membrane (Millipore, USA) using Trans-Blot Semi-dry transfer unit (Bio-Rad, USA). The membranes were blocked using 5% of bovine serum albumin (BSA) (Sigma-Aldrich, USA) for 1 h at RT. Thereafter, NC membranes were incubated overnight at 4°C with their respective primary antibodies: serotransferrin, talin-1, α-2HS glycoprotein, TTR ,and fibrinogen-α chain with 1 : 4000 dilution and with horseradish peroxidase- (HRP-) conjugated anti-mouse secondary antibody (Jackson, USA), at RT for 1 h with 1 : 8000 dilutions on the gentle shaking condition. Each membrane was then developed with enhanced chemiluminescence (ECL) (Thermo Scientific, Pierce, USA), a highly sensitive substrate detector using the Chemi-Doc_TM MP Imaging system (Bio-Rad, USA). After each incubation, membranes were washed thrice with wash buffer (0.05% tween-20 in a phosphate buffer saline).

The cell lysate was prepared from RA control and E2-treated RA-FLS through lysis in Radio-Immunoprecipitation Assay (RIPA) buffer (SIGMA) for Western blotting. Protein concentration was measured using BCA assay (G-biosciences) [45 (link)]. The 20 µg protein was resolved on 10% SDS-PAGE gel and then transferred to a nitrocellulose (NC) membrane at 20 V for 50 min using a semi-dry transfer unit (Bio-Rad, Hercules, CA, USA) followed by overnight blocking at 4 °C with 5% bovine serum albumin (BSA) in 1X Tris-buffered saline-tween 20 (TBST) [44 (link)]. The membrane was then incubated with primary antibodies STAT1 (9172T, Cell Signaling), Phospho-STAT1 (Tyr701) (9167L, Cell Signaling) (dilution 1:5000) overnight at 4 °C followed by washing and incubation with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (dilution 1:8000) (Jackson, West Grove, PA, USA) for 1 h at room temperature. The band intensity was measured by chemiluminescence (ECL) reagent (Thermo Scientific, Rockford, IL, USA). The band intensity was normalized by the housekeeping protein (β-actin; sc-47778) as a loading control.

E10.5 embryos were fixed in 4% paraformaldehyde in PBS at 4°C, rinsed in PBS, then permeabilized in PBS with 0.5% Triton X-100 for 24 h at 4°C. Neurofilament immunodetection was performed as previously described (Ray et al., 2020 (link)). Primary anti-neurofilament antibody (Developmental Studies Hybridoma Bank, 2H3) was used at a 1:20 dilution, and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch, 115-035-003) was used at 1:1000 dilution. Signal was developed using an ImmPACT DAB Substrate Kit (Vector Laboratories, SK-4105). Photographs were taken using a Nikon SMZ-U dissecting scope fitted with a Jenoptik ProgRes C5 camera.