Goat anti rabbit igg

Paraffin-embedded sections were deparaffinized, rehydrated, then steamed for 30 min in citrate buffer for antigen retrieval, after blocking 5% goat serum in Tris-buffered saline with 0.05% Tween 20. Sections were incubated with primary antibodies against either Ki-67 (1:100, ThermoFisher Scientific, Waltham, MA, USA) or Phospho-S6 Ribosomal Protein (1:400, Cell Signaling Technology). Horse-radish peroxidase-conjugated secondary antibodies (rabbit anti-goat IgG 1:200, Abcam, Cambridge, MA, USA, and goat anti-rabbit IgG, 1:100: Abcam) followed by the SignalStain DAB Substrate Kit (Cell Signaling Technology) were used for detection. Nuclear counterstaining was performed using hematoxylin. For apoptosis detection, ileal sections were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using the In Situ Cell Death Detection Kit, TMR red (Roche Diagnostics, Indianapolis, IN, USA), per manufacturer’s instructions. Sections were mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI (4′,6-diamidino-2-phenylindole) as a nuclear stain. Slides were visualized with the C1 laser scanning Confocal Microscope System (Nikon, Melville, NY, USA), and analyzed using ImageJ.

The rhKU_Sej_LRR_2271 and LipL32 (50 µg/mL) were applied to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) using a paintbrush. The membranes were dried at room temperature for 30 min, blocked with 3% BSA in phosphate-buffered saline, pH 7.4 (PBS) for 30 min, and washed twice with PBS (pH 7.4) containing 0.05% Tween 20 (PBST). Then, the membranes were incubated with each rabbit hyperimmune sera against Leptospira spp. (Table 1) and the rabbit control serum at a dilution of 1:100 for 1.5 h and washed with PBST. The membranes were incubated with goat anti-rabbit IgG (Abcam, Cambridge, UK) conjugated with gold nanoparticles (Kestrel Bioscience, Pathumthani, TH) at a dilution of 1:20 for 30 min and washed with distilled water before observing the red lines on the membranes. The rhKU_Sej_LRR_2271 was also preliminarily tested with dog plasma samples (6 positive and 2 negative samples, confirmed by the MAT, LipL32 ELISA, and rrs real-time PCR assay). The protocol was modified from the rabbit hyperimmune serum assay using goat anti-dog IgG (Abcam, Cambridge, UK) conjugated with gold nanoparticles instead of goat anti-rabbit IgG. The antibody ratio to the volume of colloidal gold was 0.01 mg of antibody to 1 mL of colloidal gold.

CIF staining with stromal markers such as α-SMA and FSP-1 was used to confirm the expression of NOX4 in GBC stroma. The above IHC samples were permeabilized in PBS containing 10% methanol for 30 min, washed in PBS, and sealed with PBS containing 3% FBS for 1 h. The M.O.M kit (Vector Laboratories, Inc., USA) was used to block Mouse IgG according to the manufacturer’s instruction. For CIF staining of NOX4 and α-SMA or FSP1, the sections were respectively incubated with rabbit anti-NOX4 (1:500; GeneTex, USA) and mouse anti-α-SMA (1:200; Abcam, UK) or mouse anti-FSP-1 (1:100; Abcam, UK) at 4 °C overnight. Then, the sections were incubated with corresponding secondary antibodies, goat anti-rabbit IgG (1:1000; Abcam, UK) for detecting NOX4 expression, goat anti-mouse IgG (1:200; Abcam) for α-SMA, goat anti-rabbit IgG (1:200; Abcam) for FSP1. Finally, the sections were washed in PBS and stained with diamidine phenylindole (DAPI) for 5 min, and observed under an immunofluorescence microscope.