Two different HNPGL cases were analyzed for immunohistochemistry (IHC). Sections were deparaffinized by using xylene and graded ethanol and rehydrated. Slides were then immersed in 10mM sodium citrate buffer, pH 6.1, and processed for the antigen-retrieval procedure, using a microwave oven operated at 720W, for 10 min. After cooling, slides were transferred to phosphate buffer saline (PBS) containing 5% bovine serum albumin (BSA, blocking solution), for 1h at room temperature and then incubated, overnight at 4°C, with rabbit polyclonal anti-PPARα antibody (1:200, Thermo Scientific Inc., USA) diluted in blocking solution. In control sections, the primary antibody was omitted. Slides were then incubated for 1h at RT with biotinylated goat anti-rabbit IgG (1:200, Vector Laboratories, Burlingame, CA, USA), diluted in blocking solution. Immuno-complexes were revealed by means of an avidin biotin system (Vectastain Elite ABC kit, Vector Laboratories), using 3,3’-diamino-benzidine (DAB Substrate kit for Peroxidase, Vector Laboratories), as the chromogen. Slides were finally dehydrated and mounted with Eukitt (Kindler GmbH & Co., Freiburg, Germany).
Dab substrate kit for peroxidase
Manufactured by Vector Laboratories
Sourced in United States
The DAB substrate kit for peroxidase is a laboratory reagent used to detect the presence and localization of peroxidase enzyme in biological samples. The kit provides a chromogenic substrate that reacts with peroxidase to produce a brown-colored precipitate, allowing for visual identification of peroxidase-positive areas in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain elite abc kit, by Vector Laboratories (7 mentions)
Vectastain abc kit, by Vector Laboratories (5 mentions)
Anti ki67 antibody, by Thermo Fisher Scientific (3 mentions)
Vectastain abc system, by Vector Laboratories (3 mentions)
Neutral buffered formalin, by Merck Group (2 mentions)
Abc kit, by Vector Laboratories (2 mentions)
Mab2538, by R&D Systems (2 mentions)
Dm4000 b microscope, by MBF Biosciences (2 mentions)
Mbf cx9000 camera, by MBF Biosciences (2 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Axiocam digital camera, by Zeiss (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Goat anti mouse hrp secondary antibody, by Jackson ImmunoResearch (1 mentions)
Madcam1, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Ab125212, by Abcam (1 mentions)
Immpress reagent, by Vector Laboratories (1 mentions)
S 1000, by Vector Laboratories (1 mentions)
Borg decloaker solution, by Biocare Medical (1 mentions)
39 protocols using dab substrate kit for peroxidase
1
Immunohistochemical Analysis of PPAR-α in HNPGL
2
Immunohistochemical Analysis of Pdx1 Expression
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Paraffin sections, 10 μm thick, were collected on polylysine glass slides [56 (link)].
The incubation with the primary antibodies for Pdx1 (ab47267 rabbit) was performed overnight at 4 °C in histoblock solution (3% BSA, 5% NGS, 20 mM, MgCl2, 0.3% Tween 20 in PBS, pH 7.2); staining procedures and chromogenic reactions were carried out according to the protocols of the Vectastain ABC kit and “DAB substrate kit for peroxidase” (Vector Laboratories, Burlingame, CA, USA). Images were obtained using an Axioplan2 microscope equipped with an Axiocam digital camera (Zeiss, Oberkochen, Germany) and processed using the Axion Vision software. The digital images were assembled into composite pictures using Adobe Photoshop (Adobe Systems, San Jose, CA, USA).
The incubation with the primary antibodies for Pdx1 (ab47267 rabbit) was performed overnight at 4 °C in histoblock solution (3% BSA, 5% NGS, 20 mM, MgCl2, 0.3% Tween 20 in PBS, pH 7.2); staining procedures and chromogenic reactions were carried out according to the protocols of the Vectastain ABC kit and “DAB substrate kit for peroxidase” (Vector Laboratories, Burlingame, CA, USA). Images were obtained using an Axioplan2 microscope equipped with an Axiocam digital camera (Zeiss, Oberkochen, Germany) and processed using the Axion Vision software. The digital images were assembled into composite pictures using Adobe Photoshop (Adobe Systems, San Jose, CA, USA).
3
Xenograft Tumor Histological Analysis
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Xenograft tumors were dissected and fixed in 10% neutral-buffered formalin (Sigma-Aldrich) and subsequently embedded in paraffin. Five-micrometer-thick sections were prepared and stained with hematoxylin and eosin (H&E). Immunohistochemistry was performed on formalin-fixed paraffin tumor sections, as previously described [47 (link)]. Primary antibodies used were anti-Ki-67 antibody (dilution 1:300; Thermo Scientific, Fremont, CA; #RB-9043-P0) and anti-cleaved caspase-3 antibodies (1:300 dilution; Cell Signaling, Cat #: 9661). Staining was developed with 3,30 diaminobenzidine (DAB) using the DAB substrate kit for peroxidase (Vector Laboratories, Burlingame, CA, SK-4100). For quantitative analysis Ki-67 or cleaved caspase-3 positive cells were counted by using NIH Image J software version 1.47 (Wayne Rasband, National Institutes of Health, Bethesda, MD).
4
Immunohistochemical Analysis of Keratin2e and Madcam1
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For the keratin2e immunostaining, sections were deparaffinised and citrate-treated in 6mM sodium-citrate buffer (pH 6). The blocking was done with 5% goat serum in 3% BSA in PBS. The samples were incubated overnight with a primary mouse antibody against keratin2e (10R-C166a, 1:200, Fitzgerald) followed by a goat anti-mouse-HRP secondary antibody (1:500; Jackson Immuno Research). Detection was done with the Vectastain Elite ABC Kit (Vector Laboratories) and counterstain with haematoxylin. The whole mount immunostaining for Madcam1 was performed with a primary rat antibody against Madcam1 (550556, 1:25; BD Pharmingen) and a secondary anti-rat-HRP antibody (1:200, Santa Cruz Biotechnology). The DAB substrate kit for peroxidase (Vector Laboratories) was used for detection. Unspecific staining was blocked with 1% dry milk in 1xPBS/0.1% Tween-20.
5
Immunohistochemistry of Brain Sections
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Immunohistochemistry procedures were performed as described previously with minor modifications13 (link),64 (link). Briefly, fresh frozen brains were sectioned at 20 µm thickness, mounted on Superfrost Plus slides (Menzel, Braunschweig, Germany) and fixed in 4% paraformaldehyde (Sigma-Aldrich, Vienna, Austria) in PBS. Endogenous peroxidases were quenched with 0.3% H2O2 in methanol and 10% normal goat serum in antibody diluent (v/v) was used for protein blocking. Sections were incubated with the primary antibodies rabbit anti-Iba-1 (1:1000) (Wako Chemicals, Neuss, Germany) or rabbit anti-CD68 (1:1000) (ab125212, abcam, Cambridge, UK) in antibody diluent (AD) (0.1 M PBS containing 0.05% Tween 20, 2% normal goat serum and 1% bovine serum albumin) overnight at 4 °C and for 30 min in AD containing the biotinylated secondary antibody (goat anti-rabbit IgG 1:200) (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, USA) at room temperature. After incubation with avidin-biotin complex solution (Vectastain Elite ABC Kit), sections were developed with 3, 3-diaminobenzidine (DAB substrate kit for peroxidase, Vector Laboratories).
6
Immunohistochemistry of Cardiac Tissue
7
Immunohistochemical Detection of PCNA
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Paraffin slices (1 μm) were dewaxed and sequentially incubated in citrate buffer (pH 6.0) for 30 min, H2O2 (3%) solution for 30 min, blocking solution (5% BSA and 0.5% Tween 20 in PBS) for 1.5 h, and finally avidine/biotin blocking (Vector Laboratories, Burlingame, CA, USA) solution for 30 min. Subsequently, the slices were incubated with the primary antibody overnight at 4 °C. After three washings with PBS, 30 min incubation with the secondary antibody at room temperature followed. After three washing steps with PBS, the ABC-kit (only used for biotinylated secondary antibody) and DAB substrate kit for peroxidase (both from Vector Laboratories) were used according to the manufacturer’s manuals. The respective primary and secondary antibodies used and their dilutions are listed in Table 2 .
To detect proliferating cells, the percentage amount of PCNA-positive nuclei was estimated by counting the number of stained nuclei in randomly defined microscopic areas and dividing by the total number of nuclei.
To detect proliferating cells, the percentage amount of PCNA-positive nuclei was estimated by counting the number of stained nuclei in randomly defined microscopic areas and dividing by the total number of nuclei.
8
Immunohistochemical Analysis of GPR1, CCRL2, and Chemerin in Rat and Human Testes
9
Immunohistochemical Analysis of Apoptosis and Myelin Markers
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Vibratome sections were used for immunohistochemistry for cleaved caspase-3 and O4. Sections were pretreated with 3% H2O2 in phosphate-buffered saline (PBS) for 15 min at room temperature followed by a PBS wash. Then, sections were incubated with 5% goat or horse serum in PBS at room temperature for 30 min, and were incubated with primary antibodies, polyclonal anti-cleaved caspase-3 antibody (1:1000, Cell Signaling Technologies, Beverly, MA) or monoclonal anti-O4 antibody (1:200, Chemicon, Temecula, CA), overnight at 4 °C. Biotinylated secondary antibodies (1:500, Vector Laboratory, Burlingame, CA) were applied for 2 h and sections were serially stained with avidin–biotin using ABC elite kit (Vector Laboratory, Burlingame, CA). Reaction products were visualized using DAB substrate kit for peroxidase (Vector Laboratory, Burlingame, CA).
Immunohistochemistry for 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) (1:500, Sigma, St. Louis, MO) and glial fibrillary acidic protein (GFAP) (1:1000, DAKO, Glostrup, Denmark) was performed on paraffin sections. After deparaffinization, rehydration, and H2O2 blocking, sections were microwaved in 10 mM citrate buffer for 5 min and were stained using the avidin–biotin complex method as described above.
Immunohistochemistry for 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) (1:500, Sigma, St. Louis, MO) and glial fibrillary acidic protein (GFAP) (1:1000, DAKO, Glostrup, Denmark) was performed on paraffin sections. After deparaffinization, rehydration, and H2O2 blocking, sections were microwaved in 10 mM citrate buffer for 5 min and were stained using the avidin–biotin complex method as described above.
10
Placental Chemerin Expression Analysis
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The sections of term placentas of normal pregnancy and PE patients were subsequently de-waxed, rehydrated, the antigen was retrieved by using a 1× Universal HIER antigen retrieval reagent (ab208572, Abcam, MA 02453, United States) and incubated with 3% H2O2. Sections were then blocked with 10% goat serum (ab7481, Abcam) for 1 h at room temperature. The sections were incubated in chemerin (1: 100, ab72965, Abcam) overnight in the cold room. Chemerin staining was visualized using a DAB substrate kit for peroxidase from Vector Laboratories and counterstained and mounted as described above. Digital photographs at 4× and 10× were taken, and Image J software was used to quantify the H-score.
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