Vectastain elite avidin biotin complex kit

Sections from control (n = 9), LPS + hyperoxia (n = 6) and LPS + hyperoxia + hAEC (n = 7) pups were exposed to heat-induced epitope retrieval using citrate buffer (pH 6) and blocked in 2% (w/v) bovine serum albumin in phosphate-buffered saline for 1 h and primary antibody applied overnight at 4 °C in a humid chamber. Immunohistochemistry was performed on paraffin-embedded sections to detect microglia (ionized calcium-binding adaptor molecule 1 (Iba1), 1:1000, 019-19741; Wako Pure Chemical Industries, Osaka, Japan), activated microglia (CD68, 1:100, MCA1957; ABD Serotec, Oxford, UK) and astrocytes (glial fibrillary acidic protein (GFAP), 1:1000, M7240; DAKO, CA, USA), as described previously [37 (link)]. hAEC cell tracking was performed using anti-human HLA-G (HLA-G clone 87G, 1:100, ALX-805-719-c100; Enzo Lifesciences, Australia). Biotinylated secondary antibodies (1:200) were applied for 1 h at room temperature in a humid chamber and reacted with the VectaStain Elite avidin–biotin complex kit (1:200; Vector Laboratories, CA, USA) using 3,3′-diaminobenzidine as the chromagen. Following counterstaining with haematoxylin, slides were coverslipped with DPX (Sigma-Aldrich, MO, USA). Omission of the primary antibody resulted in no specific staining. For each antibody, all sections were stained simultaneously to ensure uniform conditions for subsequent analysis.

Immunohistochemistry was done on formalin-fixed, paraffin-embedded, 3 μm lymphoma and normal lymph node sections using the VectaStain Elite Avidin-Biotin Complex Kit (Vector Laboratories, Inc.) as directed by the manufacturer. Sections were probed with anti-VCP mouse monoclonal antibody (Abcam Inc., catalog number ab11433, dilution 1:4000) as directed by the manufacturer, except blocking was done with 5 % normal serum in TBST for 1 h at room temperature and incubation with the secondary antibody (biotinylated anti-mouse reagent, Vector Laboratories, Inc., dilution 1:500) was done for 30 min. Staining was done using 3,3’-diaminobenzidine peroxidase substrate kit (Vector Laboratories, Inc.) as directed by the manufacturer. Negative controls were done using the primary antibody described above that was pre-incubated overnight at 4 °C with VCP peptide (792–806, Abcam Inc., catalog number ab39788) in a 1:10 antibody:peptide ratio.