Immunohistochemistry (IHC) was used to detect the presence of Ki67. Briefly, sections were dewaxed with xylene and rehydrated using decreasing concentrations of ethanol and ddH2O. Slides were washed in Tris Buffered Saline (TBS: 0.1 M Tris pH 7.5 and 0.3 M NaCl) with 0.05% Tween-20 (TBST). Endogenous peroxidase was quenched by washing with 3% H2O2 (Fisher Scientific, Loughborough, United Kingdom) in Phosphate Buffered Saline (PBS) for 5 min. Sections were blocked with 1.5% normal goat serum (Vectastain ABC Kit, Vector Labs, Peterborough, United Kingdom) in TBS for 1 h and then incubated with rabbit anti-Ki-67 antibody (ab66155; Abcam, Cambridge, United Kingdom) at 1:100 overnight at 4°C; control sections were incubated with normal goat serum 1.5%. After 3 washes with TBST, sections were incubated with biotinylated anti-rabbit IgG secondary antibody (Vectastain ABC Elite Kit, Vector Laboratories) for 1 h, followed by ABC solution (Vectastain ABC Elite Kit, Vector Laboratories) for 30 min. A 3,3′-Diaminobenzidine (DAB) peroxidase substrate kit (Vectastain ABC Elite Kit, Vector Laboratories) was used to develop the stain. The slides were counterstained with haematoxylin, dehydrated, mounted with DEPEX (VWR, Leicestershire, United Kingdom) and imaged. For analysis of Ki67 staining, GCs were classified as either Ki67 negative or positive using an assessment scale (see results).
Vectastain elite abc kit
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom, Canada, Germany, France, Japan, Switzerland
The Vectastain Elite ABC kit is a specialized laboratory equipment used for the detection and visualization of target proteins or antigens in biological samples. It utilizes an avidin-biotin complex (ABC) system to amplify the signal, enabling researchers to achieve high sensitivity and consistent results in their immunohistochemical or immunocytochemical analyses.
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Lab products found in correlation
Dab peroxidase substrate kit, by Vector Laboratories (54 mentions)
3 3 diaminobenzidine (dab), by Merck Group (34 mentions)
Bx51 microscope, by Olympus (31 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (28 mentions)
Immpact dab, by Vector Laboratories (26 mentions)
Avidin biotin blocking kit, by Vector Laboratories (24 mentions)
Diaminobenzidine substrate kit, by Vector Laboratories (20 mentions)
Dab substrate kit, by Vector Laboratories (18 mentions)
Biotinylated secondary antibody, by Vector Laboratories (18 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (16 mentions)
Vector novared, by Vector Laboratories (15 mentions)
Nis elements software, by Nikon (14 mentions)
Cleaved caspase 3, by Cell Signaling Technology (14 mentions)
Hematoxylin, by Merck Group (13 mentions)
Dab substrate, by Vector Laboratories (13 mentions)
Permount, by Thermo Fisher Scientific (13 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (12 mentions)
Ba 1000, by Vector Laboratories (12 mentions)
Target retrieval solution, by Agilent Technologies (12 mentions)
Biotinylated goat anti rabbit secondary antibody, by Vector Laboratories (11 mentions)
1 332 protocols using vectastain elite abc kit
1
Immunohistochemical Detection of Ki67 in Tissue
2
Quantifying Angiogenesis in Lung Tissue
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Immunohistochemistry was used to assess the effect of the peptides T3, T7 and LF15 on the number of blood vessels present in lung tissue. Briefly, slides were treated with peroxidase blocking reagent (DakoCytomation) for 30 mins then washed in x2 changes of dH2O. Slides were then blocked with blocking serum (VECTASTAIN Elite ABC KIT, Vector laboratories). Primary antibody (goat anti-mouse CD105, R&D Systems) or isotype control at a concentration of 15µg/ml was added to each section and incubated for 30 mins at room temp. After washing with x2 changes of T-PBS, slides were incubated with biotinylated secondary antibody (VECTASTAIN Elite ABC KIT, Vector laboratories). Slides were washed in T-PBS and incubated with Vectastain elite ABC reagent (VECTASTAIN Elite ABC KIT, Vector laboratories) for 30 mins. Next, slides were washed in T-PBS and incubated with DAB (DakoCytomation) for 10 mins. Slides were washed with dH2O, dehydrated and mounted.
3
Immunohistochemical Detection of ADRP in Liver Sections
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Paraffin-embedded liver sections (4 mm) were incubated with anti-ADRP polyclonal antibody (clone GP40-mN1, Fitzgerald Industries International, Concord, MA, USA). The immunoreaction was detected with the avidin–biotin–peroxidase complex (ABC) method [31 (link)] using ABC Vectastain-Elite Kit (Vectastain ABC Elite Kit, Vector Labs, Burlingame, CA, USA), as described previously [30 (link)].
4
Immunohistochemical Assessment of Skin Innervation
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Free‐floating bright‐field immunohistochemistry was performed for skin innervation, as previously described.23 For each patient, four serial 50 μm thickness sections, 200 μm apart, were incubated in pan‐axonal marker mouse antiprotein gene product 9.5 (PGP9.5) (1:1000, BIO‐RAD, Raleigh, NC, USA) overnight. The sections were washed and incubated in horse anti‐mouse IgG (1:50, Vectastain Elite ABC kits, Burlingame, CA, USA) followed by Avidin/Biotinylated HRP (1:50, Vectastain Elite ABC kits, USA) incubation, and were colored with sweat glands (SG) peroxidase substrate reaction (Vector SG kits, USA). The intraepidermal nerve fiber density (IENFD) was counted as the number of fibers crossing the dermal‐epidermal junction per mm with high magnification light microscopy (400×; Nikon ECLIPSE Ni‐U, Tokyo, Japan), according to a previous protocol.23
5
Immunohistochemistry Staining Protocol
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Horseradish peroxidase staining was performed using the Vectastain Elite ABC kits (Vector Laboratories, Burlingame, CA, United States), according to manufacturer’s protocols. All antibodies used are listed in Supplementary Table 3 . Antigen retrieval was performed via trypsin digestion (0.1% trypsin for 20 min at 37°C, followed by washes in deionized H2O and PBS). Primary antibody was incubated overnight at 4°C, followed by incubation in 0.3% hydrogen peroxide in methanol for 30 min at room temperature and 3 × 10 min washes in 1× PBS, to inactivate endogenous peroxidases. A 1 h biotin-conjugated secondary antibodies incubation, at room temperature was followed by a 30 min incubation with ABC mixture solution containing biotinylated HRP (Vectastain Elite ABC kits, Vector Laboratories, Burlingame, CA, United States) at room temperature, and finally the color was developed using DAB (3,3′-diaminobenzidine) solution (DAB Peroxidase substrate, Vector Laboratories). Stained sections were dehydrated through ascending ethanol solutions, mounted using DPX Mounting Medium (Thermo Fisher Scientific), and imaged using the Zeiss Axioplan 2IE Mot Microscope System (Carl Zeiss) and the Axiovision software (Carl Zeiss).
6
Immunohistochemistry Protocol for ALDH1A1
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Before dewaxing, the tissue sections were placed in a 60°C baking oven for 20 minutes. Slides were deparaffinized in xylene, rehydrated in a graded alcohol series, and washed in PBS twice for five minutes each time. Sections were heated in 10 mM sodium citrate buffer, pH = 6.0, for 15 minutes in a 95°C water bath for antigen retrieval. Until the buffer cooled down, we performed five-minute PBS washes. Endogenous peroxidase activity was blocked by incubating the sections in 3% H2O2 at room temperature for ten minutes. Blocking serum was added dropwise at room temperature for 20 minutes to reduce the non-specific background. Anti-ALDH1A1 monoclonal antibodies (ab-134188; 1:100 dilution; Abcam, Cambridge, MA, USA) were added and incubated overnight at 4°C. The sections were washed in PBS three times for two minutes, and subsequently incubated with biotinylated secondary antibody (PK-4001; VECTASTAIN® Elite ABC kits, Vector Labs, USA) for 30 minutes at room temperature. The slides were subsequently incubated with ABC (PK-4001; VECTASTAIN® Elite ABC kits, Vector Labs, USA) for another 30 minutes, washed in PBS, and stained with DAB (3, 3-diaminobenzidine). Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted.
7
Immunohistochemical Staining Protocol
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The immunohistochemical staining procedure started with the incubation in 1% hydrogen peroxidase (in PBS:Methanol 1:1) followed by 3 times of 15 min washing with PBST and 30 min incubation in 7% normal horse serum in 0.02% PBST (blocking solution). Sections were incubated ON with the desired primary antibody in blocking solution at 4 °C. The next day, sections were washed 3 times for 15 min with 0.02% PBST, followed by incubation with the secondary biotinylated antibody for 30 min (Vectastain Elite ABC Kit, Vector laboratories). After three 15 min washing steps with 0.02% PBST and incubation for 30 min in an avidin-biotin complex solution (Vectastain Elite ABC Kit), sections were washed two times for 15 min in 0.02% PBST and twice for 15 min in PBS before stained with 3,3′-diaminobenzidine tetra hydrochloride (DAB) solution (DAB substrate Kit for Peroxidase, Vector laboratories, Burlingame, USA). Staining reactions were terminated upon visual inspection by adding and washing with water. Sections were finally air-dried and mounted with Mowiol. Bright field colored images were captured with an automated Leica DM4000 B microscope coupled to a MBF CX9000 camera (MBF Bioscience) and displayed with PictureFrame™ software.
8
Immunohistochemical Staining Protocol
9
Immunohistochemical Analysis of NF-κB and COX-2
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After incubation in normal horse serum for 60 min to prevent nonspecific binding, the skin sections were incubated with mouse anti-nuclear factor (NF)-κB (sc-109, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-COX-2 (18-7379, 1:200; Zymed, USA) in phosphate buffered saline-Tween (PBS-T overnight at 4℃. The sections were subsequently incubated with biotinylated horse anti-mouse IgG (VECTASTAIN Elite ABC Kit; Vector Laboratories, USA). The immunoreactivity was assessed using the avidin–biotin peroxidase complex (VECTASTAIN Elite ABC Kit; Vector Laboratories). The peroxidase reaction was developed using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories). As a control, the primary antibodies were omitted from the immunohistochemical analysis of a few test sections in each experiment. The sections were then counterstained with hematoxylin before being mounted.
10
Immunohistochemical Detection of c-Fos
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Free-floating sections were rinsed in PBS with 0.1% IgG-free bovine serum albumin (Jackson ImmunoResearch, West Grove, PA, USA) wash buffer and then incubated in a polyclonal antibody to c-Fos primary (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-52) in wash buffer with 0.3% Triton-X100 (Sigma-Aldrich) at room temperature for 48 h at 4°C. After rinsing in wash buffer, sections were incubated for 60 min at room temperature in biotinylated anti-rabbit IgG secondary antibody (1:600; Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA, USA), rinsed in wash buffer, and then incubated in an avidin-biotin horseradish peroxidase complex (1:200, Vectastain Elite ABC kit) for 60 min at room temperature. The sections were then rinsed in wash buffer and reacted in a 0.1 M Tris buffer (pH = 7.6) with 0.56 mM 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich) solution, 0.003% hydrogen peroxide and 63 mM nickel ammonium sulfate (Sigma-Aldrich). After 5 min, sections were rinsed in Tris buffer to stop the chromagen reaction. Immunostained sections were mounted onto glass slides (Adhesion Superfrost Plus Microscope Slides, Brain Research Laboratories, Newton, MA, USA), cover slipped with DPX mounting media (Sigma-Aldrich), and imaged using bright field microscopy.
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