Vectastatin abc kit

Primary tumors and lungs from mice were fixed with 4% paraformaldehyde, paraffin-embedded, and cut into 4-μm section slices. The slides were incubated in a dry oven at 60 °C for 1 h, de-paraffinized, re-hydrated, and incubated in a citrate buffer (Dako) at 121 °C for 10 min to retrieve antigen. To block non-specific signals. the slides were incubated with 3% H₂O₂ for 15 min and with 2% horse serum in BSA solution for 1 h. The slides were incubated overnight at 4 °C with anti-KHK (1:100, Santa Cruz), anti-LRRC59 (1:100, Novus Biologicals), or anti-E cadherin (1:200, Thermo Fisher Scientific). The sections were biotinylated with a secondary antibody for 1 h. The immune complexes were visualized using the Vectastatin ABC kit (Vector Laboratories) and the DAB detection kit (Dako). Finally, the slides were counterstained with hematoxylin for 15 min, and photographed at four high-power fields in each slide.

After incubation in 3% H₂O₂ followed by blocking in 10% normal goat serum (Dako Corporation, Carpentaria, CA) in PBS, the sections were immunostained overnight at 4°C with antibodies against von Willebrand factor (vWF, dilution 1∶200; Dako), platelet endothelial cell adhesion molecule-1 (PECAM-1; dilution 1∶50, Santa Cruz Biotechnology, Santa Cruz, CA), proliferating cell nuclear antigen (PCNA; dilution 1∶200, Dako), CD34 antibody (dilution 1∶100; Abcam, Cambridge, MA). The sections were then treated with secondary antibodies (Vector Laboratories, Burlingame, CA). Immunoreactivity was visualized using the avidin-biotin complex method (Vectastatin ABC kit, Vector Laboratories) and developed with diaminobenzidine, followed by counterstaining with Gill hematoxylin (Sigma-Aldrich, St. Louis, MO).

The surgical samples were fixed in 10% phosphate-buffered formalin, embedded in paraffin, cut into 3 μm slices, and mounted on glass slides. Specimens were then incubated in 0.01 M sodium citrate (pH 6.0) for 10 min, 3% hydrogen peroxide for 30 min, and 1% bovine serum albumin for 30 min. Sections were then incubated in a humidified chamber at 4 °C overnight with BHLHE41 rabbit polyclonal antibody (1:150 dilution), stained with diaminobenzidine tetrahydrochloride (DAKO, Glostrup, Denmark) using the avidin–biotin complex and immunoperoxidase method (Vectastatin ABC Kit, Vector Laboratories, Burlingame, CA, USA), and counterstained with hematoxylin. BHLHE41 expression was determined by counting the number of cancer cells in which the nucleus was stained with the anti-BHLHE41 antibody. Evaluation of IHC was independently carried out by two board-certified pathologists (TH and AT), and the inconsistent cases were reevaluated under agreement. Ten fields within the tumors were selected and staining in 1000 cancer cell nuclei (100 cells per field) was observed at ×200 magnification using microscopy. The samples were graded as BHLHE41-positive if more than 25% of cancer cells were stained and as BHLHE41-negative if <25% of cancer cells were stained.

For immunocytochemistry (ICC), cells incubated on coverslips were fixed by 10% neutral buffered formalin (Biosesang, Seongnam, Korea). After then, the cell was permeabilized and incubated with anti-phospho-histone H2A.X (Ser139) antibody (Merck Millipore, Darmstadt, Germany) or anti-Rad51 antibody (Abcam, Cambridge, UK) at 4 °C overnight. For IF, paraffin sections of xenograft tumors were used. The slides were incubated with the Chk1 antibody (Abcam) or anti-phospho-histone H2A.X (Ser139) antibody (Merck Millipore) at 4 °C overnight. For immunostaining microscopy, images were visualized by confocal laser scanning microscope (LSM 700, Carl Zeiss, Oberkochen, Germany), and each image was captured with identical exposure settings. For immunohistochemistry, staining with cleaved caspase-3 antibody (Cell Signaling Technology, Beverly, MA, USA) was performed using Vectastatin ABC kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instruction. Staining was visualized by NovaRED substrate (Vector Laboratories), and counterstaining was performed with hematoxylin.

The ileum or distal colon was flushed with PBS and fixed in 10% buffered formalin. Immunofluorescence staining was performed as described previously41 (link). Briefly, rehydrated paraffin or frozen sections were blocked using 5% normal goat serum or M.O.M mouse Ig blocking reagent (Vector Lab) and treated with primary antibody at 4 °C overnight. The sections were further incubated with secondary antibodies conjugated with Alexa 488 (Life Technologies), counterstained with DAPI and visualized with a LSM 510 Meta confocal microscope (Carl Zeiss). For immunohistochemistry, re-hydrated paraffin sections were treated with 3% H₂O₂ in deionized water and immunostaining was performed using Vectastatin ABC kit (Vector Lab) according to the manufacturer’s protocols. Antigen-antibody complexes were detected with a DAB peroxidase kit (Dako). For Tff3 immunohistochemistry, a sample of the distal colon with a fecal pellet was collected and fixed in methanol-Carnoy’s solution overnight38 (link). The sources of the primary antibodies are in the Supplementary Materials.

It was tested on brain sections of transgenic and control mice of CRND8 strain44 (link). To unequivocally identify the fragment(s) of the peptide responsible for binding to plaques we used three separate segments: Biot-Aβ1-6_WT(D), Biot-Aβ1-6_A2V(D) and Biot-TAT(D). Seven-micrometre thick brain sections fixed in Carnoy and embedded in paraffin were used for staining. The immunoreaction was revealed by incubation with the avidin-biotin complex (Vectastatin ABC Kit, Vector Laboratories) and diaminobenzidine (DAB) as the substrate for horseradish peroxidase.