Dab chromagen

Necropsy was performed on all subjects. Tissue samples of all major organs were collected for histopathologic and immunohistochemical examination, immersion-fixed in 10% neutral buffered formalin, and processed for histopathology as previously described (20 (link)). For immuno-histochemistry, specific anti-NiV immunoreactivity was detected using an anti-NiV N protein rabbit primary antibody at a 1:5000 dilution (12 ). In brief, tissue sections were processed for immunohistochemistry using the Dako Autostainer. Secondary antibody used was biotinylated goat anti-rabbit IgG (Vector Laboratories) at 1:200 followed by Dako LSAB2 streptavidin–horseradish peroxidase. Slides were developed with Dako DAB chromagen and counterstained with hematoxylin. Nonimmune rabbit IgG was used as a negative control.

Human normal and OA cartilage sections were a kind gift from Martin Lotz (Scripps Research Institute, La Jolla, CA). Human cartilage sections and mid-coronal sections from 12-month old wild-type and Mif−/− mouse knee joints were immunostained with anti-MIF (Life Technologies). Sections were de-paraffinized and rehydrated in serial ethanol washes followed by antigen retrieval in citrate buffer. Sections were first blocked with 3% H₂O₂ (Fisher Scientific) and then with Protein Block (Dako). The primary antibody was diluted in antibody diluent (Dako) and incubated on the sections overnight at 4°C. The following day, the sections were incubated with HRP-linked secondary antibody (Dako) and developed with the DAB chromagen (Dako). The sections were counterstained in Mayer’s Hematoxylin (Sigma) and then dehydrated in serial ethanol washes.

Kidney sections were deparaffinized and rehydrated, followed by antigen retrieval in citrate buffer (pH 6) at 90–95 °C for 10 min. The slides were then washed 3 times in wash buffer (0.05% TWEEN20 in PBS), and blocked with Dual Endogenous Enzyme Block (Dako, Carpinteria, CA) for 20 min at room temperature. Slides were then washed once and incubated with 5% goat serum +2% BSA in PBS for 30 min at 37 °C. After one wash, sections were incubated with anti-CD3 antibody (Seratec, Göttingen, Germany) diluted 1:500 overnight at 4 °C. The next day, slides were washed three times, and incubated with a biotinylated goat anti-rat IgG antibody (1:250) (Southern Biotech) for 1 h at 37 °C, followed by 3 washes and a 30 min incubation at room temperature in streptavidin-HRP (Thermo-scientific) diluted 1:5000. After three washes, slides were developed with DAB-chromagen (Dako), washed, counterstained in Mayer's hematoxylin, and mounted with permount.

IHC staining of breast cancer tissue was performed for ERRβ. Five micron sections from formalin fixed paraffin embedded tissues were de-paraffinized with xylenes and rehydrated through a graded alcohol series. Heat induced epitope retrieval (HIER) was performed by immersing the tissue sections in Target Retrieval Solution, Low pH (DAKO) in the PT Link (DAKO). IHC staining was performed using the VectaStain Kit from Vector Labs according to manufacturer’s instructions. Briefly, slides were treated with 3% hydrogen peroxide, avidin/biotin blocking, and 10% normal goat serum and independently exposed to primary antibodies for ERRβ2- cl .07, 1:150, 1:240 (R&D systems, #PP-H6707-00) and ERRβsf- cl .05, 1:150 (R&D systems, #PP-H6705-00) for 1 hour at room temperature. Slides were exposed to appropriate biotin-conjugated secondary antibodies (Vector Labs), Vectastain ABC reagent and DAB chromagen (Dako). Slides were counterstained with Hematoxylin (Fisher, Harris Modified Hematoxylin), blued in 1% ammonium hydroxide, dehydrated, and mounted with Acrymount. Control tissues with the primary antibody omitted were used as negative controls. Images of the full TMA slide stained for of Hematoxylin and eosin, ERRβsf- cl .05, and ERRβ2- cl .07 are available on figshare (https://doi.org/10.6084/m9.figshare.9992891.v1).