Sections 3-µm thick were used for immunostaining. Following deparaffinization, sections were treated in Histofine® Antigen Activation Liquid (pH 9.0; Nichirei Biosciences, Inc., Tokyo, Japan) at 121°C for 15 min. Endogenous peroxidase was blocked in 0.3% hydrogen peroxide and methanol for 30 min. Sections were then incubated with antibodies for PDIA3 (catalog no. ab13506; dilution, 1:150; Abcam, Tokyo, Japan) and Ki-67 (MIB1; catalog no. M7240; dilution, 1:100; Dako Japan Co., Ltd., Tokyo, Japan) in phosphate-buffered saline containing 1% bovine serum albumin (Sigma-Aldrich Japan K.K., Tokyo, Japan) for 16 h at 4°C. The sections were further incubated with the Histofine Simple Stain™ MAX-PO (R; Nichirei Biosciences, Inc.) for 30 min, and peroxidase activity was visualized by 3,3′-diaminobenzidine. The sections were then counterstained with hematoxylin.
Histofine simple stain max po r
Manufactured by Nichirei Biosciences
Sourced in Japan
Histofine Simple Stain MAX PO (R) is a laboratory reagent used in immunohistochemical staining procedures. It functions as a detection system for the visualization of antigen-antibody reactions in tissue sections.
Automatically generated - may contain errors
Lab products found in correlation
Simple stain dab solution, by Nichirei Biosciences (3 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Real target retrieval solution, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Ab19027, by Abcam (1 mentions)
Nb500 170, by Novus Biologicals (1 mentions)
Bz x700 confocal microscope, by Keyence (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Antigen retrieval solution ph 9.0, by Nichirei Biosciences (1 mentions)
Pascal heat induced target retrieval system, by Agilent Technologies (1 mentions)
Bz 9000 microscope, by Keyence (1 mentions)
Anti mcherry ab167453, by Abcam (1 mentions)
Goat anti rabbit igg alexa 546, by Thermo Fisher Scientific (1 mentions)
Tsa cyanine 5 system, by PerkinElmer (1 mentions)
A11035, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Anti rage antibody, by Santa Cruz Biotechnology (1 mentions)
Cellsens version 1, by Olympus (1 mentions)
Sc 8316, by Santa Cruz Biotechnology (1 mentions)
22 protocols using histofine simple stain max po r
1
Immunohistochemical Evaluation of PDIA3 and Ki-67
2
Immunohistochemical Analysis of Bovine CyPA
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Mammary gland tissues collected from cattle with (n = 3) and without (n = 3) mastitis were fixed in 4% (w/v) of paraformaldehyde (PFA) or periodate lysine paraformaldehyde (PLP) overnight at 4 °C, and embedded in paraffin. Tissue sections (3 μm) were first subjected to antigen retrieval using a REAL™ target retrieval solution (DAKO). After blocking with 3% (v/v) of normal goat serum (Vector), the sections were stained with 1 μg/mL of rabbit anti-mouse/rat/human CyPA antibody (Abcam, Catalog No: ab41684, hereafter called the commercial anti-CyPA antibody), which is predicted to react to bovine CyPA according to the manufacturer’s data sheet, or 1 μg/mL of control rabbit immunoglobulin (Dako). The sections were then stained with Histofine® Simple Stain MAX PO (R) (Nichirei Biosciences) and the signal was developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB). Finally, counterstaining with hematoxylin was performed.
3
Immunohistochemical Detection of Hsp22 Expression
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Unstained sections were deparaffinized and rehydrated prior to antigen retrieval. Antigen retrieval was performed in 10 mmol/L citrate buffer (pH 6.0) in a microwave oven. Sections were incubated with anti-Hsp22 antibody for 30 min at room temperature. After washing in PBS, the sections were incubated with peroxidase-conjugated universal immune-enzyme polymer, anti-rabbit solution (Histofine Simple Stain MAX PO-R Nichirei Biosciences, Tokyo, Japan), and then visualized with 3,3-diaminobenzidine (Sigma–Aldrich) and counterstained with hematoxylin.
4
Immunohistochemical Evaluation of Hepatocyte Proliferation
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The livers were fixed in 10% buffered formalin and embedded in paraffin. Immunohistochemical staining for PCNA were used to evaluate hepatocyte proliferation in the liver as previously described (18 (link)). Briefly, 4-µm thick sections of the livers were deparaffinized and treated with 3% hydrogen peroxide to inactivate endogenous peroxidases. The sections were heated in 0.1 M citrate buffer (pH 6.0) using the autoclave. The sections were treated with 10% goat serum (Invitrogen, Carlsbad, CA, USA) for 60 min to prevent non-specific antibody binding and then incubated with anti-PCNA (1:2,000, catalog no. LS-B14132, LSBio, Seattle, WA, USA) overnight at 4°C. After washing, primary antibody-stained sections were incubated with horseradish peroxidase (HRP)-conjugated rabbit immunoglobulin antibody solution (1:1, Histofine Simple Stain MAX-PO(R), Nichirei, Tokyo, Japan), followed by 3,3′-diaminobenzidine staining (catalog no. K3468, DAKO, Tokyo, Japan). Finally, the sections were counterstained using Mayer's hematoxylin.
5
Immunohistochemical Analysis of EXOSC4 in Pancreatic Cancer
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Patient specimens from surgically resected pancreatic cancers were examined using a protocol approved by the Institutional Review Board of the Asahikawa Medical University (# 17002). Protein expression was examined by immunohistochemistry using the anti-EXOSC4 antibody (1:300) on paraffin-embedded sections (4-µm thickness). Antigen retrieval was performed using retrieval buffer (pH 9.0) (Nichirei Biosciences, Tokyo, Japan) and autoclaving at 110 °C for 10 min. Primary antibody reactions were carried out for 1 h at room temperature. Histofine Simple Stain MAX PO (R) (Nichirei Biosciences) was used as instructed by the manufacturer. Visualization of immune reactions was made using 3,3′-diaminobenzidine tetrahydrochloride solution mixed with 0.025% hydrogen peroxide. Nuclei were counterstained with hematoxylin.
6
Evaluating Bone Regeneration Markers
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Immunohistochemical assessment was performed at 2 weeks and 3 weeks after the fracture (n=5 in each group). We assessed Ki67 expression of chondrocyte within bony callus because it is one of the best-known proliferation markers.11 12 (link) We also evaluated cathepsin K expression as an osteoclast marker.13 14 (link) The sections were incubated overnight at 4℃ with anti-Ki67 antibody (1:50 dilution, NB500-170, Novus Biologicals, Centennial, Colorado, USA) or anticathepsin K antibody (1:50 dilution, ab19027, Abcam, Cambridge, Massachusetts, USA) and subsequently treated with peroxidase-labeled antimouse immunoglobulin (Histofine Simple Stain MAX PO (R), Nichirei Bioscience, Tokyo, Japan) at room temperature for 60 min. The signal was developed as a brown reaction product using the peroxidase substrate 3,3’-diaminobenzidine (Histofine Simple Stain 3,3′-Diaminobenzidine (DAB) Solution, Nichirei Bioscience). The sections were counterstained with hematoxylin and examined with a BZ-X700 confocal microscope (Keyence Corporation, Osaka, Japan). Immunopositive cells were counted in four random fields under a high-power field.
All morphometric studies of immunofluorescence and immunohistochemical staining were performed by two blinded orthopedic surgeons.
All morphometric studies of immunofluorescence and immunohistochemical staining were performed by two blinded orthopedic surgeons.
7
Immunohistochemical Detection of Phospho-MET
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Formalin-fixed, paraffin-embedded tissue Sects. (4-μm thick) obtained from in vivo experiments were deparaffinized and heated in Antigen Retrieval Citrate Solution, pH 6 (Dako, Glostrup, Denmark). The tissue sections were treated with Protein Block Serum-Free (Dako) and then incubated with a phospho-MET antibody (#3077) (Cell Signaling Technology) and then Histofine Simple Stain MAX PO(R) (Nichirei Bioscience, Tokyo, Japan). Peroxidase activity was visualized via the DAB reaction using a reagent from Dako. The sections were counterstained with hematoxylin.
8
Quantification of Lung Inflammation
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The lungs obtained from mice were fixed with 10% formalin for 1 d. Mice immunized with VLP and challenged with influenza viruses were euthanized on the last day of observation or on the first day that extreme body weight reduction was observed (<75% of initial body weight). In mice immunized with PBS and challenged with PRH1, a portion of the mice died without extreme reduction in their weight. The lungs of those mice were also collected. Their fixed lungs were paraffin-embedded, and subsequently deparaffinized in xylene and treated with 0.3% hydrogen peroxide in 100% methanol for more than 10 min to quench the endogenous peroxidase activity. Sections were heated at 95°C for 40 min in antigen retrieval solution, pH 9.0 (Nichirei Biosciences Inc.), followed by two rinses with PBS. Antibodies against MPO and Iba1 were used for immunostaining, which are neutrophil and macrophage markers, respectively. Histofine Simple Stain MAX PO(R) (Nichirei Biosciences Inc.) and Simple Stain DAB solution (Nichirei Biosciences Inc.) were used for staining. Tissues were also stained with hematoxylin and eosin at the Advanced Research Facilities and Services, Hamamatsu University School of Medicine.
9
Immunohistochemistry of ARHGEF9 in Mouse Brain
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Young adult mice (P40) were perfused transcardially with saline and then with phosphate-buffered 10% formalin. Brains were removed and processed for paraffin embedding. Paraffinized 3 μm-sections were made and mounted on slide glasses. Endogenous peroxidase activity in sections was blocked by incubation in 3% hydrogen peroxide for 5 min. Antigen retrieval was performed by the Pascal heat-induced target retrieval system (DAKO Tokyo, Japan) with 10 mM citrate buffer (pH 6.0). After treatment with 10 mM phosphate-buffered saline (PBS/pH 7.4) containing 2% BSA for 60 min, the slides were incubated with anti-ARHGEF9 (dilution at 1:100 in 2% BSA/PBS) overnight at 4°C. ARHGEF9 was then detected with immunoperoxidase polymer reagents Histofine Simple Stain MAX-PO (R) (424142, Nichirei Bioscience, Tokyo, Japan) for 30 min. The peroxidase binding sites were detected by staining with 3,3'-diaminobenzidine in 50 mM Tris (pH 7.6). Counterstaining was performed using Mayer’s hematoxylin. Images were captured with BZ-9000 microscope (Keyence, Osaka, Japan).
10
Whole-Mount Immunostaining of Zebrafish Embryos
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Embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 2–3 h at room temperature or overnight at 4 °C. Whole-mount antibody staining was performed using the following antibodies: anti-DeltaA (18D2, ZIRC) (1:50); anti-DeltaC (zdc2, Abcam, UK) (1:500); anti-DeltaD (zdd2, Abcam, UK) (1:500); anti-Scl (ref. 43 (link)) (1:50); anti-mCherry (ab167453, Abcam, UK) (1:500); and anti-RFP (8D6, MBL, Japan) (1:500). Goat anti-mouse IgG Alexa568 (A-11031, Thermo Fisher Scientific, USA) (1:1,000) and goat anti-rabbit IgG Alexa546 (A-11035, Thermo Fisher Scientific, USA) (1:1,000) were used as the secondary antibodies. The Histofine Simple Stain MAX PO (R) (Nichirei Bioscience, Japan) and the TSA Cyanine 5 System (PerkinElmer, USA) were also used for Scl detection.
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