N histofine simple stain max po multi

Subcutaneous tumors were recovered from necropsy and measured for weight and size. Half of the tumor was fixed in 10% formalin and half fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (for TEM analysis, see below). The tissue that had been fixed in 10% formalin was embedded in paraffin blocks, sliced (5 μm) and placed on glass slides. Sections were stained with Hematoxylin and Eosin (H&E). The histological examination was performed by a pathologist (E.P.). Antibodies against phosphorylated histone H2A.X (at Ser139) and activated caspase-3 (Cell Signaling, catalog number 9661) were used to determine cellular apoptosis. An antibody against γH2AX, clone JBW301 (Merck Millipore, Darmstadt, Germany) was used to determine cellular senescence. Antigen retrieval was performed in a microwave in EDTA solution (pH 8, Invitrogen). Horseradish peroxidase (HRP)-conjugated secondary antibodies were used with detection reagent N-Histofine simple stain MAX PO (MULTI) (Nichirei Biosciences). 3,3′-Diaminobenzidine (DAB; Lab Vision) was used as a chromogen.

Tumor tissues were harvested and fixed in 10% neutral buffered formalin. After paraffin embedding, tumor specimens were cut into 5-μm sections and stained with hematoxylin and eosin. NLRP1, IL-1β, Notch1, Ki-67, and caspase-3 staining were performed following the standard protocols using mouse anti-NLRP1 (ALX-804-803, 1:200; Enzo Life Sciences), Goat anti-IL-1β (#AF-201-NA, 1:200; R&D Systems), goat anti-Notch1 (sc-6014, 1:200; Santa Cruz Biotechnology), rabbit anti-Ki-67 (ab15580, 1:500; Abcam, Cambridge, MA, USA), and rabbit anti-caspase-3 (#9662, 1:1,000; Cell Signaling Technology), respectively, diluted in Dako antibody diluent (Dako North America, Carpinteria, CA, USA), followed by N-Histofine Simple Stain MAX PO (Multi) (#414152F; Nichirei Biosciences, Tokyo, Japan). Sections were counterstained with hematoxylin and reviewed by two observers.

Immunohistochemical staining was performed as reported previously with slight modification [26 (link),27 (link),28 (link)]. Surgically obtained samples were fixed in 10% buffered formalin for 24 h and embedded in paraffin. The formalin-fixed paraffin-embedded tissues were cut into 4 μm-thick sections. TROP2 antigen was retrieved using Heat Processor Solution pH 9 (Nichirei Biosciences, Tokyo, Japan) via heating at 100 °C for 40 min. The sections were incubated with rabbit anti-human TROP2 (1:1000, ab214488; Abcam, Cambridge, UK) for 90 min at room temperature followed by incubation with N-Histofine Simple Stain MAX-PO MULTI (724152; Nichirei Biosciences) for 30 min at room temperature. Immunoreactions were detected using 3,3′-diaminobenzidine tetrahydrochloride (725191; Nichirei Biosciences) as a chromogen (exposure for 10 min), and specimens were counterstained using hematoxylin.

Deparaffinization, endogenous peroxidase inactivation, and antigen retrieval of formalin-fixed, paraffin-embedded clinical tissues and immunostaining of Brm were performed as described previously17 (link)60 (link). For CD44 (#3570; Cell Signaling), MET (ab51067; Abcam), and CAV1 (sc-894; Santa Cruz) immunostaining, the sections were incubated overnight at room temperature with the corresponding antibody used for western blotting and washed in phosphate-buffered saline. N-Histofine® Simple Stain™ MAX PO (MULTI) (414154F; Nichirei Biosciences Inc.) were then applied to the slides for 30 min at room temperature, followed by three washes in PBS. The reaction products were visualized using a 50 mg/dL 3,3′-diaminobenzidine tetrahydrochloride solution containing 0.003% H₂O₂. The immunostained sections were evaluated independently by two pathologists in conjunction with hematoxylin and eosin-stained sections from the same lesions.

Microvessels in the rat hippocampus were colorized using the polyclonal rabbit anti-laminin antibody (dilution 1:1000; Dako, Glostrup, Denmark, No. Z009701) with negative immunohistochemistry controls. Laminin is a marker for basal lamina present in all microvessels, including capillaries, venules, and arterioles. The sections were deparaffinized in xylene, rehydrated and successively treated with cooled acetone for 10 min; Proteinase K for 6 min at room temperature; 5% normal goat serum for 20 min at room temperature; primary antibody solution for 36 h at 4°C; 50% N-Histofine Simple Stain MAX PO (Multi, Nichirei Biosciences Inc., Tokyo, Japan) for 30 min at room temperature; and, colorized for 1–4 min in liquid diaminobenzidine (DAB) Substrate Chromogen System (Dako, DAB Chromogen). Immunostained sections were counterstained with Gill’s hematoxylin. In the final step, sections were dehydrated in an alcohol series cleared by xylene, treated with mounting medium and coverslipped.

Human paraffin embedded tissue arrays were purchased from US Biomax, Inc. (Rockville, MD, USA). Breast cancer and adjacent normal tissue array (BR724), and breast carcinoma with breast tissue microarray (BC08116d) were used. The tissue arrays were incubated with anti-SET antibody (1:800) for 40 min at 37 °C after blocking with Protein Block (DAKO, Tokyo, Japan), and the secondary antibody (N-Histofine simple stain MAX-PO (Multi), Nichirei Bioscience Inc. Tokyo, Japan) was applied as described previously^{39 (link)}. The stained tissues were observed and photographed at equivalent to 40 × using NanoZoomer-XR and NDP.view2 (Hamamatsu Photonics K.K., Shizuoka, Japan). The relative staining intensity of SET in breast tissues was reviewed by two independent individuals. The score was calculated based on the staining intensity as percentage of stained cells × intensity score.