Briefly, all fresh tissues were immersed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, USA) for 30 min at room temperature; and we performed ethanol-gradient dehydration, paraffin embedding, sectioning at 6 μm, and dewaxing in xylene. Tissue sections were blocked with immunohistochemical blocking solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China) at 37°C for 30 min. We discarded the blocking solution, added the immunohistochemical cleaning solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China), and washed the sections three times at room temperature for 5 min each. The primary antibody was added and the sections incubated at 37°C for 45 min. We discarded the antibody, added the immunohistochemical cleaning solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China), and washed the sections at room temperature three times for 5 min each. The secondary antibody was then added, and sections were incubated at 37°C for 45 min. The antibody was discarded, we added the immunohistochemical cleaning solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China), and then washed the sections at room temperature three times for 5 min each. Finally, we added the immunofluorescence blocking solution (Sigma-Aldrich, St. Louis, USA) for mounting.
Immunofluorescence blocking solution
Manufactured by Merck Group
Sourced in United States
Immunofluorescence blocking solution is a laboratory reagent used to reduce non-specific binding in immunofluorescence assays. It is designed to block background staining and improve the signal-to-noise ratio in these types of experiments.
Automatically generated - may contain errors
Lab products found in correlation
Immunohistochemical blocking solution, by Beyotime (5 mentions)
Paraformaldehyde (pfa), by Merck Group (5 mentions)
Immunohistochemical cleaning solution, by Beyotime (3 mentions)
Immunohistochemical washing solution, by Beyotime (3 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscopy, by Nikon (1 mentions)
6 protocols using immunofluorescence blocking solution
1
Immunohistochemical Staining Protocol
2
Immunohistochemical Analysis of Tissue Samples
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Briefly, fresh tissues were immersed in 4% paraformaldehyde (Sigma-Aldrich) for fixation at room temperature for 30 min. The tissues were then dehydrated in an ethanol gradient, embedded in paraffin, sectioned (thickness: 6 μm), and immersed in xylene for dewaxing. Tissue sections were blocked with immunohistochemical blocking solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China) at 37 °C for 30 min. The blocking solution was then discarded, and the sections were washed three times at room temperature for 5 min each with immunohistochemical washing solution (Beyotime Biotechnology). Then, primary antibodies (Table 1 ) were added and incubated at 37 °C for 45 min. After incubation, the antibody solution was discarded, and the sections were washed three times at room temperature for 5 min each with immunohistochemical washing solution (Beyotime Biotechnology). Then, secondary antibodies (Table 1 ) were added and the tissues were incubated at 37 °C for 45 min. After incubation, the antibody solution was discarded, and the sections were washed three times at room temperature for 5 min each with immunohistochemical washing solution (Beyotime Biotechnology). Finally, immunofluorescence blocking solution (Sigma-Aldrich) was added, and the sections were mounted.
3
Immunofluorescence Staining of Tissue Sections
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Briefly, fresh tissues were immersed in 4% paraformaldehyde (Sigma-Aldrich) for fixation at room temperature for 30 min. The tissues were then dehydrated in an ethanol gradient, embedded in paraffin, sectioned (thickness: 6 μm), and immersed in xylene for dewaxing. Tissue sections were blocked with immunohistochemical blocking solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China) at 37 °C for 30 min. The blocking solution was then discarded, and the sections were washed three times at room temperature for 5 min each with immunohistochemical washing solution (Beyotime Biotechnology). Then, primary antibodies were added and incubated at 37 °C for 45 min. After incubation, the antibody solution was discarded, and the sections were washed three times at room temperature for 5 min each with immunohistochemical washing solution (Beyotime Biotechnology). Then, secondary antibodies were added and the tissues were incubated at 37 °C for 45 min. After incubation, the antibody solution was discarded, and the sections were washed three times at room temperature for 5 min each with immunohistochemical washing solution (Beyotime Biotechnology). Finally, immunofluorescence blocking solution (Sigma-Aldrich) was added, and the sections were mounted.
4
Immunohistochemical Staining Protocol
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Briefly, all fresh tissues were soaked in 4% paraformaldehyde (SigmaAldrich, St. Louis, USA) at room temperature and fixed for 30 min. Ethanol gradient dehydration, paraffin embedding, slicing (6μm thickness), and soaking in xylene for dewaxing were performed. The tissue sections were blocked with an immunohistochemical blocking solution (BeyotimeBiotechnology Co., Ltd., Zhejiang, China) at 37°C for 30 min. We discardedthe blocking solution, added immunohistochemical cleaning solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China), and washed at room temperature for 5 min three times. Then, the primary antibody (Table 1 ) was added and incubated at 37°C for 45 min. We discarded the antibody, added immunohistochemical cleaning solution (BeyotimeBiotechnology Co., Ltd., Zhejiang, China), and washed for 5 min at room temperature three times. Then, the secondary antibody (Table 1 ) was added and incubated at 37°C for 45 min. We discarded the antibody, added immunohistochemical cleaning solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China), and washed for 5 min at room temperature three times. Finally, an immunofluorescence blocking solution (Sigma Aldrich, St. Louis, USA) was added to seal the film.
5
Immunohistochemical Protocol for Tissue Sections
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All fresh tissues were immersed in 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 30 min. Ethanol gradient dehydration, paraffin embedding, slicing (thickness = 6 μm ), and foaming in xylene dewaxing were carried out. The tissue sections were blocked with immunohistochemical blocking solution (Beyotime Biotechnology Co., Ltd.) at 37 °C for 30 min. The blocking solution was discarded and the immunohistochemical cleaning solution (Beyotime Biotechnology Co., Ltd.) was used to wash the sections for 5 min at room temperature three times. Then, primary antibodies were added and incubated at 37 °C for 45 minutes. The antibody was discarded, and the immunohistochemical cleaning solution (Beyotime Biotechnology Co., Ltd.) was used to wash the sections for 5 min at room temperature three times. Then, the secondary antibody was added and incubated at 37 °C for 45 min. The antibody was discarded, and the immunohistochemical cleaning solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China) was used to wash the sections for 5 min at room temperature three times. Finally, immunofluorescence blocking solution (Sigma-Aldrich) was added.
6
Immunofluorescence Staining of NLRC5 and PD-L1
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HEC-1A and Ishikawa cells were permeabilized for 20 min with 0.1% Triton X-100
(Thermo Fisher Scientific) after being fixed for 30 min with 4%
paraformaldehyde. Samples were then stained for 45 min at 37 °C with primary
anti-NLRC5 and anti-PD-L1 antibodies, rinsed (5 min/wash at room temperature)
with an immunohistochemical washing solution (Beyotime), and stained with
appropriate secondary antibodies for 45 min at 37 °C. Immunofluorescence
blocking solution (Sigma) was applied after 3 further washes as described above,
and slices were mounted and photographed using laser scanning confocal
microscopy (Nikon).
(Thermo Fisher Scientific) after being fixed for 30 min with 4%
paraformaldehyde. Samples were then stained for 45 min at 37 °C with primary
anti-NLRC5 and anti-PD-L1 antibodies, rinsed (5 min/wash at room temperature)
with an immunohistochemical washing solution (Beyotime), and stained with
appropriate secondary antibodies for 45 min at 37 °C. Immunofluorescence
blocking solution (Sigma) was applied after 3 further washes as described above,
and slices were mounted and photographed using laser scanning confocal
microscopy (Nikon).
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