The paraffin blocks were cut to 5 μm thickness, dewaxed in xylene, and rehydrated 7 times for 5 min each with descending graded alcohol. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase for 20 min. After washing in PBS, the sections were each incubated with polyclonal rabbit anti-TRPV1 (NB 100-1617; Novus Biologicals, Centennial, CO, USA; diluted 1:250 in PBS) and IL-31RA (bs-2631R, Bioss Antibodies, Boston, MA, USA; diluted 1:300 in PBS) at 4 °C for 2 days with the appropriate humidity. A biotinylated antibody (Goat Anti-Rabbit IgG Antibody Biotinylated, Vector Laboratories, Burlingame, CA, USA) was applied as the secondary antibody. The standard ABC staining method was carried out in the subsequent process. Between steps, sections were washed thrice with 0.01 M PBS for 10 min. Slides were analyzed under a microscope.
Nb100 1617
Manufactured by Novus Biologicals
Sourced in United States
The NB100-1617 is a laboratory equipment product from Novus Biologicals. It is designed to perform a specific function within the laboratory setting. The core functionality of this product is to facilitate a particular aspect of research or analysis, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (1 mentions)
Pvdf film, by Merck Group (1 mentions)
Nb120 16007, by Novus Biologicals (1 mentions)
A0265, by ABclonal (1 mentions)
Anti pi3k, by ABclonal (1 mentions)
Gb 0032, by Keygen Biotech (1 mentions)
A19056, by ABclonal (1 mentions)
St506, by Beyotime (1 mentions)
Mab5416, by Merck Group (1 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Ab1504, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
4 protocols using nb100 1617
1
Immunohistochemical Analysis of TRPV1 and IL-31RA
2
TRPV1 Receptor Immunofluorescence Assay
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The IEC-6/TRPV1+ cells were cultured on coverslips in 24-well plates overnight. Briefly, cells were stimulated with NOVO (10 μM), SB-705498 (0.1 μM) for 24 h. At the end of incubation, cells were fixed with 4% paraformaldehyde in 0.1 M PBS for 20 min. The cells were then treated by 0.25% (v/v) TritonX-100 for 5 min, and washed again. Followed by blocking in 5% (v/v) horse serum for 60 min, cells were then incubated with primary antibodies for the anti-TRPV1 receptor antibody (1:1000, NB100-1617, Novus) overnight in a 4°C refrigerator. Alexa Flour® 488-conjugated goat anti-rabbit IgG (Zhongshan Goldenbridge Biotechnology) at 1:100 dilution was used as the secondary antibody that incubated cells for 60 min. Thereafter, the coverslips were mounted face down onto slides with mounting medium containing DAPI and stored at 4°C before microscopy. Fluorescence images were captured using a fluorescence microscope (Olympus, Japan).
3
Western Blot Analysis of Cell Proteins
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Cell protein was extracted using a lysis buffer (P0013B, Beyotime, Nanjing, China), which was supplemented with protease and phosphatase inhibitor cocktail (ST506, Beyotime, Nanjing, China; GB-0032, KeyGEN, Nanjing, China). After incubation on ice for 30 min, the lysates were centrifuged at 12,000 rpm for 10 min at 4 ℃ and the supernatants were collected. The total proteins were separated by 6%-10% SDS-PAGE gels, then immediately transferred onto PVDF films (Millipore, Darmstadt, Germany). The films were furtherly probed by primary antibodies overnight (4℃), and then incubated with corresponding secondary antibodies. A BIORAD imaging system (chemiDOCTMXRS, USA) was applied to visualized the protein bands. The primary antibodies used in this study were listed as follows: Anti-TRPV1 (NB100-1617, Novus Biologicals, Colorado, USA), Anti-SR-3A (sc-390168, Santa Cruz, USA), Anti-5HT-H209 (NB120-16007, Novus Biologicals, Colorado, USA), anti-mTOR (2983, CST, Boston, USA), anti-p-mTOR (5536, CST), anti-PI3K (A0265, ABclonal, Wuhan, China) and anti-GAPDH (A19056, ABclonal, Wuhan, China).
4
Western Blot Analysis of Brain Proteins
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The brain tissues were homogenized in RIPA buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (Roche, Mannheim, Germany) on ice and stored at −80°C until use. Protein concentrations in the supernatant were determined using a BCA assay kit (Beyotime, Shanghai, China). The proteins were separated through the polyacrylamide SDS gels and transferred to 0.45 μm PVDF membrane (Millipore, Darmstadt, Germany). After blocking with 5% non-fat milk, the membranes were incubated with a primary antibody at 4°C overnight. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies (anti-mouse: 1:5,000, 7076S, Cell Signaling; anti-rabbit: 1:5,000, 7074S, Cell Signaling) after washing with TBST (TBS containing 0.2% Tween-20). Then, immunoreactivity was detected by a chemiluminescent reagent (Amersham-GE, Pittsburgh, United States). The following primary antibodies were used (dilution, source): TRPV1 (1:1,000, NB100-1617, Novus Biologicals, Littleton, CO), GAPDH (1:3,000, 2118S, Cell Signaling), GluA1 (1:1,000, AB1504, Millipore), GluA2 (1:1,000, 13607S, Cell Signaling), GluA3 (1:500, MAB5416, Millipore), Src (1:1,000, 2110S, Cell Signaling), p-Src (Tyr416) (1:1,000, 2101S, Cell Signaling), p-Src (Tyr527) (1:1,000, 2105S, Cell Signaling), cofilin (1:1,000, 5175S, Cell Signaling), and p-cofilin (1:1,000, 3313S, Cell Signaling).
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