Porcine native stomach (PNS) muscle tissue and PDSF (n = 6 each) were fixed in 10% formalin, embedded in paraffin, and sliced into 5-μm sections, which were mounted on slides. The slides were stained with hematoxylin and eosin (H&E), Masson trichrome, and 4′,6-diamidino-2-phenylindole (DAPI). For immunohistochemical (IHC) staining, the slides were placed in antigen retrieval citrate buffer (Biogenex, Fremont, CA) in a steamer for 10 min at 95 °C. Endogenous peroxidases were blocked by incubation with Peroxide Block (Innogenex, San Ramon, CA), and nonspecific binding was blocked with normal goat serum (Vector Laboratories, Burlingame, CA). Sections were incubated with primary antibodies against collagen I, laminin, MHC-I, MHC-II, and galactose-α-1,3-galactose (α-gal; all from Abcam, Cambridge, MA) overnight at 4 °C. The sections were washed, received an application of a biotinylated secondary antibody for 30 min, and were treated with streptavidin-horseradish peroxidase complex (using the Vectastain ABC Kit) and diaminobenzidine solution (both from Vector Laboratories) and counterstained with hematoxylin. The sample sections were dehydrated, mounted, and imaged using the Vectra multispectral slide analysis system (PerkinElmer, Waltham, MA).
Diaminobenzidine solution
Manufactured by Vector Laboratories
Sourced in United States
Diaminobenzidine solution is a chromogenic substrate used in immunohistochemistry and in situ hybridization techniques. It is commonly used to visualize the presence of target antigens or nucleic acid sequences in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain abc kit, by Vector Laboratories (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Antigen retrieval citrate buffer, by BioGenex (1 mentions)
Vectra multispectral slide analysis system, by PerkinElmer (1 mentions)
11βhsd1, by Cayman Chemical (1 mentions)
Sc 1004, by Santa Cruz Biotechnology (1 mentions)
Avidin biotin complex, by Vector Laboratories (1 mentions)
Avidin biotin peroxidase complex reagent, by Vector Laboratories (1 mentions)
Ab36122, by Abcam (1 mentions)
Abc solution, by Vector Laboratories (1 mentions)
Biotinylated anti goat igg, by Vector Laboratories (1 mentions)
Anti calretinin, by Santa Cruz Biotechnology (1 mentions)
Vectastain abc reagent, by Vector Laboratories (1 mentions)
Zen blue software, by Zeiss (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Ab1565, by Merck Group (1 mentions)
Vectamount, by Vector Laboratories (1 mentions)
Bloxall, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Vectashield hard set mounting medium h 1400, by Vector Laboratories (1 mentions)
8 protocols using diaminobenzidine solution
1
Histological Analysis of Porcine Stomach Tissue
2
Histological Analysis of Brain Samples
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Samples for histological examination were fixed in 4% paraformaldehyde at 4°C for 24 h before embedding in paraffin wax (Paraplast Tissue Embedding Medium, Tyco Healthcare Group, Mansfield, MA, USA). A manual microtome (Leica Microsystems, Wetzlar, Germany) was used to cut 6 µm thick coronal sections of whole brain samples. After identifying the level of the paraventricular nucleus (PVN), sections were collected for immunostaining.
After dewaxing the samples, antigens were retrieved in 10 mM citric acid just below boiling temperature for 2 h, followed by endogenous peroxidase inactivation with 3% hydrogen peroxide for 20 min. Samples were then blocked with 5% goat serum for 30 min and incubated overnight with the primary antibody for 11β-HSD1 (1:100, Cayman Chemicals) and glucocorticoid receptor (GR) (1:400, sc-1004, Santa Cruz Biotechnology). A biotinylated secondary antibody was applied together with the corresponding Avidin–Biotin-Complex and diaminobenzidine solution following the company’s protocol (all Vector Laboratories, Burlingame, CA, USA). Stained and unstained cells in the PVN were counted and the percentage of stained cells was determined.
After dewaxing the samples, antigens were retrieved in 10 mM citric acid just below boiling temperature for 2 h, followed by endogenous peroxidase inactivation with 3% hydrogen peroxide for 20 min. Samples were then blocked with 5% goat serum for 30 min and incubated overnight with the primary antibody for 11β-HSD1 (1:100, Cayman Chemicals) and glucocorticoid receptor (GR) (1:400, sc-1004, Santa Cruz Biotechnology). A biotinylated secondary antibody was applied together with the corresponding Avidin–Biotin-Complex and diaminobenzidine solution following the company’s protocol (all Vector Laboratories, Burlingame, CA, USA). Stained and unstained cells in the PVN were counted and the percentage of stained cells was determined.
3
Fixation and Immunostaining for Rat Brain
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Fixation and immunostaining procedures used in this study were previously described [24 (link)]. In brief, the rats were transcardially perfused with 70 ml of cold sodium phosphate buffered saline (PBS: 0.01 M phosphate buffer containing 0.9% sodium chloride, pH 7.2, 4°C) followed by 35 ml of 4% paraformaldehyde in PBS. The brain stem was removed, postfixed in 4% paraformaldehyde solution for 1 h, and transferred to a PBS containing 20% sucrose (pH 7.4). Frozen brain tissues were sectioned in the coronal plane (5 μm). Sections were first incubated with 0.3% H2O2 in methanol for 30 min, followed by incubation with 5% skim milk in tris-buffered saline (20 mM tris-HCl, 0.9% sodium chloride, 0.02% tween 20, pH 7.4) for 60 min. They were incubated with goat polyclonal anti-APA antibody (1 : 100, ab36122, abcam) for 120 min, biotinylated rabbit anti-goat immunoglobulin G for 120 min, and avidin-biotin-peroxidase complex reagents for 30 min and finally stained with diaminobenzidine solution for 8 min according to the manufacturer's instructions (Vector Laboratories). Each step was followed by washing the sections with PBS. Sections incubated without primary antibodies were used as negative controls.
4
Immunohistochemical Visualization of Calretinin
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Some sections were stained following typical hematoxylin and eosin protocols to observe morphology. Other sections were labeled with immunohistochemistry following typical protocols. Briefly, sections were rehydrated and treated with 3% H2O2 in dH2O to remove endogenous peroxidases. Non-specific binding was blocked with 2% bovine serum albumin and 0.4% Triton X-100 in phosphate-buffered saline (PBS) for 1 h at room temperature. Slides were incubated in a humid chamber at 4 °C for 24 h in anti-calretinin (Santa Cruz Biotechnology, Dallas, TX, USA) at 1:1000 made in blocking solution. Following PBS rinses, sections were incubated at room temperature for 1 h in biotinylated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA) at 1:100 made in blocking solution. Following PBS rinses, sections were treated with ABC solution (Vector Laboratories) for 1.5 h and exposed to diaminobenzidine solution (Vector Laboratories) until sufficient staining was observed. Sections were dehydrated and coverslipped before viewing.
5
AVP Immunohistochemistry in SON and PVN
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Three sections per animal per region (supraoptic nucleus (SON) and PVN) were stained for AVP. These were matched for bregma between different animals. Sections were washed between each step for 3 × 5 min in 0.01 M Tris-buffered saline (TBS, pH 7.4) and all steps were carried out at room temperature unless otherwise specified. Sections were blocked with BLOXALL (Vector laboratories, UK) for 10 min, then blocking solution (2% goat serum, 0.3% Triton-X in 0.01 M TBS) for 60 min, rabbit anti-AVP (1:7500 in blocking solution, AB1565, Millipore, UK) for 24 h followed by biotinylated goat-anti-rabbit (3 µg/ml in blocking solution) for 45 min, then 30 min in VECTASTAIN ABC reagent (avidin-biotinylated horseradish peroxidase complex, Vector Laboratories, UK), before developing in diaminobenzidine solution (Vector Laboratories, UK) for 5 min. Washed sections were mounted onto glass microscope slides and coverslipped with Vectamount (Vector Laboratories, UK). Slides were then imaged at 20× using an Axio Scan Z1 (Zeiss). The optical density of AVP immunoreactive cells in the supraoptic and paraventricular nuclei were quantified as grey density per area minus background in digitised images using Zen Blue software (Zeiss).
6
Multimarker Immunohistochemistry Protocol
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Immunohistochemistry was carried out on paraffin sections obtained from HDBR. Antigen retrieval consisting of boiling sections in 10 mM sodium citrate pH 6 for 10 min was used for all stains. Primary antibodies were diluted in 20% blocking serum in pH 7.6 Tris buffered saline (TBS) and sections incubated overnight at 4°C. Primary antibodies used: KID 1/5000 DAB, 1/2000 fluorescent (Invitrogen PA5–29490), KI67 1/800 (Novus Biologicals NBP2-22112), ALDOA 1/100 (Sigma HPA004177).
For colourmetric stains, sections were incubated 1 h at room temperature with biotinylated secondary antibody (1/200) followed by incubation for 1 h with ABC (Vector Labs) and developed with diaminobenzidine solution (Vector Labs), washed, counterstained with nuclear fast red, dehydrated and then mounted using DPX.
For immunofluorescence sections were incubated with secondary antibodies 1/200 1 h room temperature, counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI;ThermoFisher) and mounted with Vectashield H1400 Hardset Mounting Medium (Vector Labs). Extensive TBS washes were carried out between each step.
For colourmetric stains, sections were incubated 1 h at room temperature with biotinylated secondary antibody (1/200) followed by incubation for 1 h with ABC (Vector Labs) and developed with diaminobenzidine solution (Vector Labs), washed, counterstained with nuclear fast red, dehydrated and then mounted using DPX.
For immunofluorescence sections were incubated with secondary antibodies 1/200 1 h room temperature, counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI;ThermoFisher) and mounted with Vectashield H1400 Hardset Mounting Medium (Vector Labs). Extensive TBS washes were carried out between each step.
7
Immunohistochemical Localization of NOS3
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Immunostaining was performed on 5-μm paraffin sections with immunoperoxidase visualization. After routine deparaffinization, heat-induced epitope retrieval was performed with slides immersed in 0.01 M sodium citrate buffer (pH 6.0). To block endogenous peroxidase activity and nonspecific binding of antibody, sections were first preincubated for 1 h at room temperature in 0.1 M phosphate-buffered saline containing 10% normal goat serum and 0.3% H 2 O 2 before being incubated for 20 h at 4 ° C with rabbit polyclonal anti-NOS3 (1: 200 dilution; Bioss Inc., Boston, Mass., USA) as primary antibodies. The sections were then treated for 1 h at room temperature with biotinylated goat anti-rabbit IgG (1: 100; Jackson ImmunoResearch Laboratories Inc., West Grove, Pa., USA), followed by reacting them with the reagents from an ABC kit (avidin-biotin complex, VECTASTAIN ABC KIT, pk4000; Vector Laboratories Inc., Burlingame, Calif., USA) ac-cording to the manufacturer's recommendations; the reaction products were visualized by diaminobenzidine solution (Vector Laboratories). The sections were dehydrated by increasing concentrations of ethanol, cleared by xylene, and coverslipped with Permount (Fisher, Pittsburgh, Pa., USA).
8
Quantifying Tumor Vascularity by CD31 IHC
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Paraffin-embedded fragments of the tumour species were sectioned (5-μm thicknesses). Sections were pre-incubated with 2% bovine serum albumin in PBS for 1 h, and then, the primary anti-CD31 antibody was added and incubated for 45 min (1:50; Abcam). The sections were incubated with HRP-conjugated anti-rabbit secondary antibody (1:250; Dako, Glostrup, Denmark). After development with diaminobenzidine solution (Vector Laboratories Burlingame, CA, USA), the sections were counterstained with haematoxylin. Sections were evaluated under a microscope (Olympus BX60; Olympus, Tokyo, Japan) at 20× magnification and analysed with ImageJ software (NIH). Blood vessel density was quantified by counting the total number of CD31-positive vessels across the entire section of the tumour. For this analysis, three tumour sections were cut, one at the top, one in the middle and one at the back. Overall, five representative pictures were taken per available field.
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