Anesthetized rats were perfused intracardially with phosphate buffered saline (PBS; pH 7.4) for 10 min, followed by buffered paraformaldehyde (PFA 4%, Sigma) for 10 min. The nodose ganglia was visually identi ed and harvested from the rats and post xed by immersion in buffered PFA 4% for 12 h at 4°C followed by three 5 min washes in PBS, sucrose gradient (5%, 10%, 20%, 30% in PBS), and then embedded in optimum cutting media. Cryostat sections (20 µm) of the nodose ganglia were obtained and mounted on Superfrost Plus slides (Thermo Fisher Scienti c). Sections were blocked/permeabilized in 0.5% Triton X-100, 2% sh skin gelatin (Sigma-Aldrich), 1% bovine serum albumin in PBS for 1 h at room temperature. Sections were incubated overnight at 4°C with a mouse anti-P2X monoclonal antibody (1:100 in the same blocking media, Millipore). After being washed with PBS, tissue sections were incubated for 1 h at room temperature with an Alexa-Fluor 488 rabbit anti mouse IgG (1:200, Molecular Probes) antibody. Finally, sections were mounted in DAPI-containing media (Vectashield, Vector Laboratory) and visualized using a confocal laser microscope (Leica).
Sh skin gelatin
Manufactured by Merck Group
Sh skin gelatin is a laboratory product manufactured by Merck Group. It is a natural polymer derived from the collagen in animal skin. Sh skin gelatin is commonly used as a stabilizing agent and as a gelling agent in various laboratory applications.
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Lab products found in correlation
2 protocols using sh skin gelatin
1
Immunohistochemical Analysis of P2X Receptor
2
Immunofluorescence Staining of SETD1A and H3K4me3
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Cells were washed with phosphate buffered saline (PBS) three times and xed with 4% PFA in PBS for 15 min, then cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min, blocked with 0.2% sh skin gelatin (Sigma-Aldrich, #G7041) in PBS for 30 min at room temperature and incubated with primary antibody diluted in PBS at 4℃overnight. Next day, second antibody diluted in PBS was used to combine primary antibody at room temperature for 1h and the location was detected with ZEISS LMF880 laser scanning confocal microscope. The primary antibodies were SET1A (CST, #61702. 1:400) and H3K4me3 (abcam, #ab1012.1:200). The secondary antibody was Cy3-conjugated anti-rabbit IgG (Sigma-Aldrich, #C2306. 1:1,000). Cell nuclei were counterstained with DAPI. ). D, Fold-change of SETD1A levels in lung cancer cell lines compared to small airway epithelial cell (SAEC). Data were form previous reports12. E, Kaplan-Meier plot of cumulative overall survival of patients with lung carcinoma in the TCGA database. Patients in the 90% (SETD1A higher expression group, blue line, n = 440) were compared with patients in the lower 10% (SETD1A lower expression group, yellow line, n = 48). The P-value of the log-rank test comparing the two survival curves was 0.067. Statistical test of A-C were analyzed by using the Welch's t test after log transformation. n means independent biological sample.
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