Avidin biotin complex

GFAP with an accompanying Nissl stain was used for astrocyte quantification (Riquier & Sollars, 2020 (link)). Following PBS rinses (5 × 5 min 0.2% Triton X‐100), sections were blocked in 5% NGS for 90 min, incubated with rabbit anti‐GFAP antibody (1:13,000, Abcam, Cat. # ab7260, RRID: AB_305808), and then rinsed and blocked for 30 min in methanol containing 0.3% H₂O₂.
Following three rinses, both Iba1 and GFAP sections were placed in a goat anti‐rabbit antibody solution containing 2% NGS and 0.3% Triton X‐100 for 2 h at room temperature (1:1000, Vector Laboratories, Cat. # BA‐1000, RRID: AB_2313606). Sections were rinsed and then visualized using a 2% avidin–biotin complex solution containing 0.1% Triton X‐100 (Vector Laboratories, Burlingame, CA) for 1 h followed by a 0.05% diaminobenzidine solution containing 0.125% nickel ammonium sulfate and 0.01% H₂O₂. Every immunohistochemistry run contained negative control sections that were processed identically to experimental tissue except for omission of the primary or secondary antibody. No staining was observed on negative control tissue.

Immunohistochemical staining was undertaken using the En Vision two-step method (DAKO, Glostrup, Denmark) following the manufacturer's instruction. The formalin-fixed, paraffin-embedded tissues were sectioned and deparaffinised and rehydrated routinely. The slides were then incubated with a primary antibody, directed against WBP5 (Santa Cruz, Dallas, TX, USA; 1 : 100 dilution), at 4 °C overnight. The biotinylated secondary antibody (1 : 500 dilution; Dako, Carpinteria, CA, USA) and the avidin–biotin complex (Vector Labs, Burlingame, CA, USA) were then applied according to the manufacturer's recommendations.
All slides were assessed by two pathologists. The results were classified semiquantitatively based on the total scores of the percent positivity of stained tumour cells and the staining intensity. The percent positivity was scored as 0 if <5% (negative), 1 if 5–30% (sporadic), 2 if 30–70% (focal) and 3 if >70% (diffuse) of cells stained, whereas staining intensity was scored relative to the known positive and negative controls as 0 if no staining, 1 if weakly to moderately stained and 2 if strongly stained (Takahashi et al, 2009 (link)). The score⩾2 is regarded as ‘High' and the score <2 is regarded as ‘Low' in immunohistochemical staining.

Sections from formalin-fixed and paraffin-embedded rat lung tissue were dewaxed by incubating at 65℃ overnight and then rehydrating in continuous xylene and ethanol rinses. After incubation with hydrogen peroxide, the slides were washed with PBS and then incubated with 0.4% Triton X-100. After washing with PBS, the sections were incubated with a blocking solution (Dako Protein Block, Dako, Glostrup, Denmark) for 30 min at 37℃. The sections were incubated with primary antibodies (anti-Ki67 and anti-cleaved caspase3, both from CST, diluted 1:200) at 4℃ overnight, and washed several times with PBS and then with the corresponding biotinylated secondary antibody (diluted at 1:500). Sections were then rinsed with PBS multiple times. After adding the avidin–biotin complex (Vector Laboratories, USA), diaminobenzidine (DAB) detection reagent (Enzo Life Sciences, USA) was used to visualize the slices fixed using fixative (Vector Laboratories, Burlingame, USA).