SARS-CoV-2 IHC was performed with rabbit-polyclonal anti-SARS-CoV/SARS-CoV-2 nucleocapsid primary antibody (1:6500 dilution; Sino Biological, Wayne, PA, USA) and followed by a biotinylated donkey anti-rabbit IgG secondary antibody (1:1500 dilution; Jackson ImmunoResearch Laboratories, Inc). The biotinylated target was detected by ABC-AP kit (Vector Labs, Newark, CA, USA) using the ImmPACT Vector Red substrate and counterstained with hematoxylin. Stains were semiquantitatively scored for staining intensity and distribution as follows: 0, no staining; 1, weakly positive (fewer than 5 cells per section) and/or low intensity staining; 2, moderately positive (5–20 cells per section) and/or medium intensity staining; 3, strongly positive (more than 20 cells per section) and/or high intensity staining.
Abc ap kit
Manufactured by Vector Laboratories
Sourced in United States
The ABC-AP kit is a laboratory equipment product offered by Vector Laboratories. It is designed for a specific core function, but a detailed description while maintaining an unbiased and factual approach is not available at this time.
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Lab products found in correlation
Vector red, by Vector Laboratories (2 mentions)
Immpact vector red substrate, by Vector Laboratories (1 mentions)
Ab732, by Abcam (1 mentions)
Abc elite kit, by Vector Laboratories (1 mentions)
Rat anti cd31, by BD (1 mentions)
Vectastain abc, by Vector Laboratories (1 mentions)
Rabbit anti glut1, by Abcam (1 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Rat anti cd31, by Dianova (1 mentions)
Aec kit, by Vector Laboratories (1 mentions)
Anti cd8, by Thermo Fisher Scientific (1 mentions)
Anti pd 1, by R&D Systems (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Anti cd21, by Thermo Fisher Scientific (1 mentions)
Meca79, by Thermo Fisher Scientific (1 mentions)
Anti pax5, by Abcam (1 mentions)
Anti cd4, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Abcam (1 mentions)
Vectastain abc hrp kit, by Vector Laboratories (1 mentions)
Dako retrieval solution, by Agilent Technologies (1 mentions)
10 protocols using abc ap kit
1
SARS-CoV-2 Nucleocapsid Protein Immunohistochemistry
2
Immunostaining for E. coli-laden Macrophages
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LP macrophages with or without E. coli co-localisation were identified in mucosal biopsies using previously validated immunolabelled CD68+ [19 (link)] and E. coli antibodies [20 (link)]. A rapid indirect immunostaining protocol was employed for macrophage-specific CD68 (PG M1) to minimize risk of mRNA degradation [21 (link)]. CD68 positive cells were detected using a Vectastain ABC-AP kit and a Vector Red chromogenic substrate. Detection of intracellular E. coli was achieved by co-staining CD68+ macrophages with an anti-E. coli polyclonal antibody and labeled with Vector Blue chromogenic substrate. Staining was visualized using a Zeiss axioplan MOT 400 M microscope. CD68+ cells co-localising with anti-E. coli antibody were termed E. coli-laden macrophages (Fig. 1a, b ). CD68+ cells without anti-E. coli antibody co-localisation were termed E. coli-unladen macrophages (Fig. 1c ).![]()
CD68 staining macrophages (red) can be seen to contain E. coli ( blue ) in the biopsy from a patient with CD ((
3
Immunohistochemical Staining of Gal1 in Melanoma
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Formalin-fixed, paraffin-embedded sections were deparaffinized during 18 h in xylene followed by rehydration essentially as described (34 (link)). Heat-mediated antigen retrieval was performed using citrate-based retrieval buffer (10 mM). Samples were blocked in peroxide blocking buffer (3% H2O2), followed by blocking buffer (PBS; 1% BSA and 5% normal serum) during 1 h at room temperature. Samples were incubated with HMB45 + M2-7C10 + M2-9E3 anti-melanoma cells Ab (ab732; Abcam) or with anti-Gal1 rabbit polyclonal Ab at 4 °C overnight in a humidified chamber. Then, samples were washed and incubated with a biotinylated secondary Ab (ABC Elite Kit; Vector Laboratories) followed by streptavidin–alkaline phosphatase incubation (ABC-AP Kit; Vector Laboratories). Samples were then washed in PBS, incubated in red chromogen (Vector Red), and counterstained with Mayer’s hematoxylin. The Gal1 score was calculated by a semiquantitative assessment of both the intensity of staining (graded as 0 to 3 using adjacent normal parenchyma as reference) and the percentage of positive cells (graded as 0 to 5). Expression levels were categorized as low/medium or high according to the median value of the score.
4
Immunohistochemical and Immunofluorescence Analysis of Tumour and Lung Metastases
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After removal, tumours and lungs were fixed in 4% paraformaldehyde and then embedded in paraffin. 7 μm sections were deparaffinised with xylene and rehydrated with graded ethanol. The sections were stained according to routine immunohistochemistry procedures and visualised by Vectastain ABC or ABC-AP kit (Vector Laboratories, Burlingame, CA). Alternatively, samples were frozen in OCT and fixed in acetone/methanol before standard immunofluorescence procedures.
Primary antibodies used for immmunohistochemistry and immunofluorescence: Rat anti-CD31 at 1:200 dilution (BD Pharmingen; 550274), Rat anti-CD31 (DIANOVA; DIA-310), biotinylated mouse anti-SMA-alpha at 1:200 (Thermo Scientific; 14-9760-82), rat anti-NKp46 (BioLegend; 137606), rabbit anti-GLUT-1 (Abcam; ab652) at 1:500, rabbit anti-cleaved caspase-3 (Cell Signalling; 9661) at 1:500. The fluorochrome-conjugated Alexa 488 (A11070; A11006; A11017) and Alexa 568 (A11077; A11031) were used as secondary antibodies (1:200).
Lung metastases in the LLC model was analysed by Haematoxylin and Eosin (H&E) staining on 10 μm lung paraffin serial sections at day 14 post tumour injection26 (link).
Primary antibodies used for immmunohistochemistry and immunofluorescence: Rat anti-CD31 at 1:200 dilution (BD Pharmingen; 550274), Rat anti-CD31 (DIANOVA; DIA-310), biotinylated mouse anti-SMA-alpha at 1:200 (Thermo Scientific; 14-9760-82), rat anti-NKp46 (BioLegend; 137606), rabbit anti-GLUT-1 (Abcam; ab652) at 1:500, rabbit anti-cleaved caspase-3 (Cell Signalling; 9661) at 1:500. The fluorochrome-conjugated Alexa 488 (A11070; A11006; A11017) and Alexa 568 (A11077; A11031) were used as secondary antibodies (1:200).
Lung metastases in the LLC model was analysed by Haematoxylin and Eosin (H&E) staining on 10 μm lung paraffin serial sections at day 14 post tumour injection26 (link).
5
Paraffin Embedding and Trichrome Staining
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Liver samples were fixed in 10% buffered formalin for embedding in paraffin. For the Masson's trichrome staining, 5‐μm sections were deparaffinized, rehydrated, and stained with eosin as a counter stain. The antibodies were stained using an ABC‐AP kit and AEC kit (Vector Laboratories, Cambridgeshire, UK), according to standard procedures.
6
Immunohistochemical Analysis of Lymph Nodes
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Lymph nodes were fixed in 10% neutral-buffered formalin and embedded in paraffin. Tissue sections were typically 4-μm thick and were examined after staining with hematoxylin and eosin (H&E). For immunohistochemistry, the sections were first boiled in a citrate-based solution to retrieve antigens and subsequently quenched in 3% hydrogen peroxide. The antibodies used included anti-FOXP3 (Cat. MAB8214, R&D systems, Minneapolis, MN), anti-PD-1 (Cat. AF1021, R&D systems), anti-CD3 (Cat. ab5690, Abcam, Cambridge, MA), anti-PAX5 (Cat. sc-1974, Santa Cruz Biotechnology, Dallas, TX), anti-CD4 (Cat. 14–0042, eBioscience, San Diego, CA), anti-CD8 (Cat. 14–0081, eBioscience), anti-CD21 (Cat. Ab75985, Abcam), MECA79 (Cat. 53–6036-80, eBioscience) and anti-HA (Cat. 3724, Cell Signaling Technology) antibodies. After biotinylated secondary antibody treatment, visualization was carried out with Vectastain ABC-HRP kit (Vector Laboratories, Burlingame, CA) or ABC-AP kit (Vector Laboratories) in combination with DAB or with VectorRed (Vector Laboratories) respectively.
7
Hyaluronic Acid Immunohistochemistry
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Sections were deparaffinized and dried overnight at 37°C. Sections were incubated in a water bath at 60° C in a 1:10 diluted DAKO retrieval solution (# S1699, Dako, Glostrup, Danmark). After cooling down for 30 minutes half of the retrieval solution was replaced by distilled water. After 10 minutes sections were placed in distilled water for 5 minutes. After two washes in Tris-buffered saline (TBS; 0.05 M Tris-HCl at pH 7.6 and 0.15 M NaCl) for 5 minutes, the sections were incubated with 1% bovine serum albumin in TBS for 30 minutes (Dako). Biotinylated hyaluronic acid binding protein (HABP, # 385911 Calbiochem, Merck, Darmstadt, Germany) diluted 1:75 in Antibody Diluent (# B 1-31C, Medac, Wedel, Germany) was applied for 1 h at room temperature. The binding sites were detected using the ABC-AP-Kit (Vector Laboratories Inc., Burlingame, CA). The Permanent Red Kit (Dako) was used as a chromogen. Sections were counterstained with hemalumn (Merck, Darmstadt, Germany), dehydrated and covered with Eukitt (Kindler, Freiburg, Germany).
8
Immunohistochemical Dual Staining Protocol
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The IHC staining was done as previously described [24 (link)]. Mouse on mouse blocking reagent for vimentin staining, ABC-HRP kit and ABC-AP kit were purchased from Vector Laboratories (Burlingame, CA). CD31 antibody (Thermo Fisher Scientific, Reinach, Switzerland) and biotinylated goat anti-rabbit secondary antibody (Vector Laboratories) were diluted 1:50 and 1:200, respectively. Vimentin antibody and biotinylated goat anti-mouse secondary antibody (Vector Laboratories) were diluted 1:200 and 1:800, respectively. CYR61 antibody (Santa Cruz Biotechnology, Inc., Heidelberg, Germany, #sc13100) and biotinylated goat anti-rabbit secondary antibody were diluted 1:150 and 1:600, respectively. For CD31/vimentin double-staining, tissues were first stained with anti-CD31 and biotinylated goat anti-rabbit secondary antibody, followed by anti-vimentin-staining and biotinylated goat anti-mouse secondary antibody. Substrates used were DAB (Sigma-Aldrich) and Vector Red (Vector Laboratory).
9
Recombinant Rispens Virus Characterization
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Example 6
Growth kinetics and plaque morphology of recombinant Rispens/rpsLneo-DsRed2 and parent Rispens were compared. Briefly, 9.5×105 cells of CEF and 950 plaque forming unit of one of the recombinant Rispens/rpsLneo-DsRed2 viruses or the parent Rispens strain were planted into 6-well plates. Cells were harvested at 0, 24, 48, 72, 96, 120, or 144 hour. The cells were trypsinized and resuspended in 1 ml of LM medium, and titrated immediately by plaque assay. For plaque assay, CEF cells were infected with serial tenfold dilutions of trypsinized cells. Four days later, plaques were visualized by black plaque assay. Briefly, the cells were fixed with methanol:acetone mixture (1:2) and incubated with anti-Rispens monoclonal antibody 2BN90 (AVIAN DISEASES 37: 561-567, 1993). Next, incubated with biotinylated anti-mouse IgG antibody and then with VECTASTAIN ABC-AP kit, Rispens plaques were stained by addition of NBT/BCIP solution. The numbers of the plaques were counted macroscopically and the average size of fifty plaques was calculated using the program cellSens standard (OLYMPUS) for plaque morphology.
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10
Immunohistochemical Analysis of Femur Tissue
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Femurs were isolated and fixed in 3.7% formaldehyde (Sigma-Aldrich). Samples were decalcified with decalcifying solution (Sigma-Aldrich) and processed with a TP1020 tissue processor (Leica Biosystems, Wetzlar, Germany) to prepare paraffin blocks, and 5.0 μm thick sections were prepared. For trichrome staining, a NovaUltraTM Masson trichrome stain kit (IHC World, Woodstock, MD, USA) was used. For immunohistochemistry staining, the VECTASTAIN ABC kit or ABC-AP kit (Vector Laboratories, Burlingame, CA, USA) was used. Briefly, rehydrated samples were boiled with 0.01 M sodium citrate (Sigma-Aldrich) for antigen retrieval. For blocking activity of endogenous hydrogen peroxidase, the samples were treated with 0.5% H2O2. Next, the samples were permeabilized with 0.3% Triton X-100 (Sigma-Aldrich). For blocking nonspecific binding of antibodies, the samples were incubated with 2% normal horse serum for 1 h at room temperature (RT) and then treated with primary antibodies against CD31, endomucin. The samples were incubated with biotinylated secondary antibodies for 1 h at RT, followed by incubation with an avidin–biotin complex solution. The substrate solution, ImmPACT NovaRed or Vector Blue (Vector Laboratories), was used to visualize the reactive area in the tissue. Finally, samples were counterstained with Fast Red (Vector Laboratories).
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