Tissue sections were deparaffinized and rehydrated through xylene and graded ethanols. Slides were rinsed in water and then in phosphate buffered saline (PBS). The Vectastain Universal Kit (Vector Laboratories, Burlingame, Calif., USA) was used following the instructions supplied by the manufacturer. Briefly, sections from postnatal 6 day rat molars were incubated for 30 min in diluted blocking serum, and then incubated overnight at 4 °C with the mouse primary IgM antibody to human Stro-1 (Hybridoma Bank, Iowa City, Iowa). After rinsing in PBS for 5 min, sections were treated with prediluted biotinylated pan-specific universal secondary antibody (Goat anti-mouse IgM) for 30 min and then incubated with the streptavidin/peroxidase complex for 30 min. The immunostaining reaction was next developed with a diaminobenzidine tetrahydrochloride (DAB) substrate kit (Vector Laboratories, Burlingame, Calif., USA) for 5 min.
Vectastain universal kit
Manufactured by Vector Laboratories
Sourced in United States, Denmark
The Vectastain Universal kit is a versatile tool used in immunohistochemistry and other related techniques. It contains all the necessary components to perform a reliable and efficient detection of target antigens in biological samples. The kit provides a standardized and consistent approach to labeling and visualization, making it a valuable resource for researchers and laboratories.
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Lab products found in correlation
Diaminobenzidine tetrahydrochloride dab substrate kit, by Vector Laboratories (1 mentions)
Antigen unmasking solution, by Vector Laboratories (1 mentions)
Dab peroxidase hrp substrate kit, by Vector Laboratories (1 mentions)
Tunel staining, by Promega (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Dp72 ccd camera, by Olympus (1 mentions)
Neutral buffered formalin, by Thermo Fisher Scientific (1 mentions)
Ab56416, by Abcam (1 mentions)
Bmk 2202, by Vector Laboratories (1 mentions)
Eclipse microscope, by Olympus (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
5 protocols using vectastain universal kit
1
Immunohistochemical Detection of Stro-1 in Rat Molars
2
Immunohistochemical Staining of Lung Tissue
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Lung tissue sections were prepared by the Pathology Core of Harvard Medical School (Boston, MA) and used for immunohistochemical staining as follows: (1) Deparaffinization was done in xylene (10 min, three times), followed by 100% ethanol, 95% ethanol, 80% ethanol, and 50% ethanol (5 min, two times each). Tissue sections were then rinsed with water and 1 × PBS buffer; (2) Antigen retrieval was performed by boiling in 1 × antigen unmasking solution (Vector Laboratories, Inc., Burlingame, CA) for 30 min and allowed to cool to room temperature. Tissue sections were then washed with water and 1 × PBS; (3) Permeabilization was done by incubating slides in 0.1% Triton X-100 in 0.1% sodium citrate for 30 min on ice. The tissue sections were then stained with specific antibodies using the R.T.U. Vectastain® Universal Kit followed by the DAB Peroxidase (HRP) Substrate Kit, per the manufacturer’s instructions (Vector Laboratories, Inc.).
3
Immunohistochemical Analysis of Tumor Samples
4
Immunohistochemical Analysis of Calprotectin
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Specimens were fixed in 10% formaldehyde in PBS, and were embedded in paraffin. Five μm sections were deparaffinized with a xylene and ethanol series. Sections were incubated with a mouse monoclonal anti-human Myeloid/Histiocyte antigen (S100A8/S100A9) (calprotectin) antibody (clone MAC 387) (Dako Denmark A/S, Glostrup, Denmark) (1:200 dilution), a Vectastain Universal kit (Vector Laboratories Inc.) was used according to the manufacturer’s instructions and staining signals were visualized with diaminobenzidine. Images were recorded using an AX80 microscope equipped with a DP72 CCD camera (Olympus, Tokyo, Japan).
5
Placental Histology and Immunohistochemistry
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Placentas and fetuses were dissected by cesarean section on E14.5. Harvested samples were fixed in 10% neutral buffered formalin (Thermo Fisher Scientific) at room temperature and embedded in paraffin. At least five placentas from different dams with the indicated genotypes or treatments were sectioned and stained with hematoxylin and eosin to assess morphology. Histologic images were captured by use of a Nikon Eclipse microscope equipped with an Olympus DP71 color camera under 2×, 20×, and 40× objectives. Measurement of size and thickness of different placental layers were performed using ImageJ (National Institutes of Health).
For immunohistochemical staining of mouse placentas, deparaffinized tissues were quenched with 3% H2O2, blocked for 2 h, and incubated with primary antibodies anti-p62 (1:500; ab56416; Abcam) overnight. The M.O.M. kit (BMK-2202; Vector Laboratories) was applied for blocking and secondary antibody incubation according to the manufacturer’s instructions. The sections were then incubated with the ABC reagent from the Vectastain universal kit (PK-7200; Vector Laboratories) and developed using the DAB substrate kit (SK-4100; Vector Laboratories). Tissues were counterstained with hematoxylin. A no–primary antibody staining was included as a negative control.
For immunohistochemical staining of mouse placentas, deparaffinized tissues were quenched with 3% H2O2, blocked for 2 h, and incubated with primary antibodies anti-p62 (1:500; ab56416; Abcam) overnight. The M.O.M. kit (BMK-2202; Vector Laboratories) was applied for blocking and secondary antibody incubation according to the manufacturer’s instructions. The sections were then incubated with the ABC reagent from the Vectastain universal kit (PK-7200; Vector Laboratories) and developed using the DAB substrate kit (SK-4100; Vector Laboratories). Tissues were counterstained with hematoxylin. A no–primary antibody staining was included as a negative control.
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