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.
Immunohistochemical washing solution
Manufactured by Beyotime
Sourced in China
The Immunohistochemical washing solution is a laboratory reagent used to wash and rinse tissue samples during the immunohistochemical staining process. It helps to remove excess staining reagents and unbound antibodies from the sample, ensuring proper signal detection and reducing background noise.
Automatically generated - may contain errors
Lab products found in correlation
Immunofluorescence blocking solution, by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Immunohistochemical blocking solution, by Beyotime (2 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscopy, by Nikon (1 mentions)
Gb11124, by Wuhan Servicebio Technology (1 mentions)
Eclipse ti u, by Nikon (1 mentions)
4 protocols using immunohistochemical washing solution
1
Immunohistochemical Analysis of Tissue Samples
2
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.
3
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).
4
Immunofluorescence Staining of NLRC5 and LC3
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HEC-1A, AN3CA, Ishikawa, and their shRNA-NLRC5 cells were xed in 4% paraformaldehyde (Sigma) for 30 min, washed with phosphate buffered saline (PBS), and then permeabilized with 0.1% Triton X-100 (Thermo Fisher Scienti c) for 20 min. Subsequently, primary antibodies, anti-NLRC5 (DF13672, A nity) and anti-LC3 (GB11124, Servicebio) were added, and they were incubated at 37 °C for 45 min. After incubation, the antibody solution was discarded, and each section was washed thrice with immunohistochemical washing solution (Beyotime, Shanghai, China) at room temperature for 5 min. Next, secondary antibodies were added, and the tissues were incubated at 37 °C for 45 min. After incubation, the antibody solution was discarded, and each section was washed thrice with immunohistochemical washing solution at room temperature for 5 min. Finally, the immuno uorescence blocking solution (Sigma) was added, sections were mounted, and images were captured using a confocal laser-scanning microscope (Nikon, Eclipse Ti-U, Japan). The double immuno uorescence staining of NLRC5 (DF13672, A nity) and MHC I (ab281903, Abcam, Cambridge, MA, USA) in NLRC5 HEC-1A cell was operated as above described.
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