Histostain plus bulk kit

Cells were washed three times with HBS, fixed with methanol for 10 min at −20 °C and washed further prior to blocking with 10% normal goat serum. Visualization of binding by anticytomegalovirus primary antibody, clone 8B1.1 diluted 1:500 (Millipore, MAB810) was performed using the Histostain^®-Plus Bulk Kit followed by the AEC Single Solution kit (Invitrogen, Carlsbad, CA, USA). Colored images were taken with EVOS XL Core Imaging System (Invitrogen, Carlsbad, CA, USA) at 20× magnification and phase contrast images were taken with AMG EVOS FL Digital Inverted Microscope (Fisher Scientific, Rockford, IL, USA) at 10X magnification. IE-positive nuclei were averaged from five random fields per well and the average from three wells was determined in each experiment.

Hippocampal tissue was fixed in 10% neutral buffered formalin, dehydrated, and paraffinized at room temperature for 24h, followed by sectioning (4–5 µm). Sections were stained with hematoxylin and eosin for light microscopy analysis.
For GFAP immunohistochemistry, brain sections were incubated in 10% H₂O₂ for 30 min to eliminate endogenous peroxidase activity and blocked for one hour with 10% normal goat serum at room temperature. Sections were then incubated in primary anti-GFAP antibodies (Abcam, Cambridge, MA, USA; 1/1000) for 24 h at 4 °C. Antibody detection was performed using the Histostain-Plus Bulk kit (Invitrogen, Camarillo, CA, USA) against rabbit IgG, and finally, 3,3′-diaminobenzidine was used for visualization. A Nikon microscope was used to capture photomicrographs at 400× magnification (Eclipse E200-LED, Tokyo, Japan). GFAP expression was estimated by counting GFAP (+) cells in random sections of each rat.

The tissues were fixed by submersion in neutral-buffered formalin (Sigma-Aldrich, Schnelldorf, Germany) for 24 h, and then trimmed and placed in histological cassettes for dehydration, inclusion in paraffin (Sigma-Aldrich) and staining with hematoxylin and eosin (Merck KGaA, Darmstadt, Germany). The tissues were subjected to an indirect immunoperoxidase method as follows: Antigen retrieval was achieved by exposing the samples to 90°C for 30 min in citrate buffer (10 mM, pH 6.0; Sigma-Aldrich), followed by overnight incubation at 4°C with the mouse monoclonal anti-human mitochondria antibody (ab3298; Abcam, Cambridge, MA, USA). Next, the samples were treated using a streptavidin-biotin detection method (using a Histostain-Plus Bulk Kit, a LAB-SA Detection System and a DAB-Plus Reagent Set; Invitrogen Life Technologies, Camarillo, CA, USA) followed by hematoxylin counterstaining. The images of the tissues were obtained with a DM3000 laboratory microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA).

Staining of Kras (Abcam, #ab180772), Nras (Abcam, #ab206969), Tpr (Abcam, #ab84516), Akt3 (Abcam, #ab152157), Vimentin (Abcam, #ab92547), Hepatocyte (HepPar1) (Dako, #M7158), Ppm1d (Abcam, #ab31270), Cxcr4 (Abcam, #ab124824), Arhgef11 (Santa Cruz Biotechnology, #sc-166740), and Plekha5 (Santa Cruz Biotechnology, #sc-390311) were performed on formalin-fixed paraffin-embedded (FFPE) tissue using Histostain-Plus Bulk Kit (Invitrogen, #85-8943) according to the manufacturer’s instruction. Briefly. Paraffin-embedded mammary gland, PT, liver, or lung samples were sliced into 4 µm thickness. Next, slides were treated with xylene and followed by 100% alcohol treatment for deparaffination. After treated with peroxidase quenching solution (3% hydrogen peroxidase in methanol) for 10 min to block endogenous peroxidase, the slides were boiled in epitope retrieval buffer at 96–100 °C for 20 min for antigen retrieval. Following washes with PBS and incubation with blocking solution (Reagent A) for 10 min, slides were then incubated with primary antibody (1:100–500 dilution) for 1 h. After washed with PBS, slides were further incubated with the biotinylated second antibody (Reagent B) for 10–20 min and followed by enzyme conjugate (Reagent C) treatment for 10 min. Sections were stained with DAB and then counterstained with hematoxylin.