Rabbit anti rat igg

Formalin-fixed paraffin embedded liver sections were deparaffinized in xylene, rehydrated in graded alcohols and washed in distilled water. Antigen retrieval for neutrophil binding epitopes was performed by boiling sections in citrate buffer (10 mM, pH 6.0) for 24 min, follow by incubating the slides with trypsin (1 mg/ml) at 37 °C for 15 min. To block endogenous peroxidase activity, slides were incubated with 3% H₂O₂.methanol. Liver sections were washed and incubated with 10% BSA in PBS containing biotin (25% v/v) and avidin (25% v/v) to minimize non-specific binding of primary antibody. Slides were then incubated with primary antibody, anti-Ly6B.2 (1:200, clone: 7/4, Biorad) or isotype control (RIgG2a, 1:200) overnight at 4 °C. After washing, the sections were incubated with diluted secondary antibody (rabbit anti-rat IgG, 1:200, Vector BA4001), followed by VECTASTAIN ABC reagent (Vector PK6100). Slides were developed with chromogen diaminobenzidine (DAB, DAKO K3468) and counterstained with Gills Haematoxylin. Stained sections were digitized using Aperio Digital Pathology System and analyzed with ImageScope V9 softeware.

Aristolochic Acid I (A9451, sodium salt) was obtained from Sigma-Aldrich (Castle Hill, NSW, Australia). Primary antibodies utilised in these experiments were: rat anti-mouse Ly6G to detect neutrophils (Abcam, Melbourne, Victoria, Australia); rat anti-mouse F4/80 to detect macrophages (Bio-Rad, Gladesville, NSW, Australia); rabbit anti-CD31 to detect endothelial cells (Cell Signaling, San Diego, CA, USA); rabbit anti-cleaved caspase 3 to detect cell death (Cell Signaling), and; goat anti-collagen IV (Southern Biotechnology, Birmingham, AL, USA). Biotinylated secondary antibodies (rabbit anti-goat IgG, rabbit anti-rat IgG, or goat anti-rabbit IgG) and the VECTASTAIN Elite ABC HRP Kit for immunoperoxidase staining were obtained from Vector Laboratories (Burlingame, CA, USA).

For flow cytometry, antibodies against mouse antigens were purchased from Biolegend (San Diego, CA) or BD Biosciences (Franklin Lakes, NJ) unless otherwise specified and included CD24-PE (M1/69), CD31-B (MEC13.3), TER-119-B (TER-119), CD45-B (30-F11), CD29-FITC (HMB1-1), CD61-APC and streptavidin-APC-Cy7. For immunohistochemistry, the following antibodies were used: anti-SMA (1A4; Sigma), anti-p63 (4A4), K8/18 (Troma-1; Developmental Studies Hybridoma Bank, Iowa City), anti-Keratin 5 (Covance, Emeryville, CA), anti-Keratin 14 (LL002; Novocastra, UK), anti-E-cadherin, anti-ASAP1 (Rockland Immunochemicals Inc., Limerick, PA), anti-PROX1 (ab37128; Abcam, Cambridge, UK), anti-SNAI2 (#9585 Cell Signaling Technology, Massachussetts, USA). Secondary antibodies included biotin-conjugated goat anti-rabbit IgG, rabbit anti-rat IgG and goat anti-mouse IgG (Vector Laboratories, Burlingame, CA).

CD41 Immunohistochemistry was performed according to established protocols for assessment of the platelet amount in the myocardium [10 (link)]. Briefly, sections were incubated with a rat anti-mouse CD41 IgG1 antibody (Gene Tex, Inc., Irvine, CA, USA, Ref: GTX76011) as primary antibody and an isotype control antibody (Bio-Rad Laboratories, Watford, UK, #MCA 1211) as negative control. A biotinylated Rabbit anti-Rat IgG (Vector Laboratories, Burlingame, CA, USA, BA-4001) for secondary staining was used. For detection of the secondary antibody, Vectastain® ABC Kit (Vector Laboratories, Burlingame, CA, USA, Ref: PK-6100) with the substrate VECTOR® Red (Vector Laboratories, Burlingame, USA, Ref: SK-5100) and Levamisole (Dako North America, Inc., Carpinteria, CA, USA, Ref: X3021) was used.
Platelets were counted in two representative pictures of 20× magnification of the inflamed myocardium of two different CD41-stained slices in a non-blinded fashion, as described before [10 (link),23 (link)].

Liver tissue sections were fixed in 4% buffered formalin and embedded in paraffin or snap frozen in OCT compound. Fixed, paraffin-embedded tissue sections were incubated with antibody to F4/80 (1:100 dilution; clone BM8, eBioscience), followed by horseradish peroxidase- or alkaline phosphatase-conjugated secondary antibodies (1:100 dilution; rabbit anti rat IgG; Vector Labs). Frozen tissues were stained in ORO (Sigma-Aldrich). At least five non-overlapping fields per sample were imaged using Aperio eSlide Manager and ImageScope software (Leica Biosystems) and quantitation was performed using ImageJ software using thresholding of object sizes to eliminate debris. Average ORO droplet size was calculated by dividing the sum of stained area by the total number of droplets in the same field.