Mice were deeply anaesthetized and transcardially perfused with 20 ml PBS followed by 20 ml of cold 4% paraformaldehyde (PFA). Brains (from 3 mice in each group) were then removed and post-fixed with the same 4% PFA solution for 4–6 h at 4 ℃. The samples were then transferred to 30% sucrose in PBS overnight. Sample Sects. (14 μm) were prepared on gelatin-coated glass slide via cryosection. The sections were then washed three times in PBS for 5 min each, blocked with 5% bovine serum albumin (BSA) and 0.2% Triton X-100 for 1 h at room temperature and then incubated with primary antibody against mTOR (CST, Beverly, MA, USA) or NLRP1 (Santa Cruz, CA, USA) at 4 °C overnight. After washing with phosphate-buffered solution (PBS), samples were incubated with appropriate secondary antibody at room temperature for 1 h and cell nuclei were stained with DAPI. Fluorescent images were captured with a Pannoramic MIDI scanner (3D HISTECH, Budapest, Hungary).
Nlrp1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
NLRP1 is a protein that plays a key role in the activation of the inflammasome, a multi-protein complex involved in the inflammatory response. It functions as a sensor for detecting cellular stress and damage, and its activation leads to the production of pro-inflammatory cytokines. This product provides a tool for researchers to study the structure, function, and regulation of NLRP1 in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 1, by Santa Cruz Biotechnology (2 mentions)
Pannoramic midi scanner, by 3DHISTECH (1 mentions)
Caspase 4, by Proteintech (1 mentions)
Ripa lysis buffer strong, by CWBIO (1 mentions)
Gapdh, by CWBIO (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab42080, by Abcam (1 mentions)
Permafluor aqueous mounting medium, by Thermo Fisher Scientific (1 mentions)
A11004 alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Z1 axioobserver lsm10 confocal microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Actin, by Cell Signaling Technology (1 mentions)
Tubulin, by Cell Signaling Technology (1 mentions)
Hybond ecl membrane, by GE Healthcare (1 mentions)
Ecl reagent, by PerkinElmer (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Nlrp3, by Adipogen (1 mentions)
4 protocols using nlrp1
1
Immunohistochemical Analysis of mTOR and NLRP1
2
Western Blot Analysis of Inflammasome Proteins
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MCF7 cells transfected for 72 h and cultured in hUCMSC-CM for 24 h were lysed in 50 μL RIPA lysis buffer (strong) containing 0.5 μL phenylmethylsufonyl fluoride (CWBio, Jiangsu, China) and incubated on ice for 2 h. The lysed cells were incubated with 50 μL of 2x SDS-PAGE protein loading buffer (Bio-Rad) in boiling water for 10 min. After centrifugation, the protein samples were subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Merck Millipore). The membranes were blocked in TBST (3.0 g Tris-HCl, 8.0 g NaCl, 0.1% Tween-20, and pH 7.4) containing 5% nonfat dried milk for 1 h and incubated overnight at 4°C with diluted primary antibodies against GAPDH (1 : 2000; CWBio, Jiangsu, China), caspase-1 (1 : 500; Santa Cruz Biotechnology, Delaware, USA), NLRP1 (1 : 100; Santa Cruz Biotechnology), caspase-4 (1 : 500; Proteintech, Wuhan, China), and ASC (1 : 1000; Proteintech, Wuhan, China). The membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1 : 2000; CWBio, Jiangsu, China) at 20–25°C for 1 h and then with ECL substrate solution (1 : 1 (v/v); CWBio, Jiangsu, China). The bands were quantified using Photoshop 7.0, and the gray value ratio of bands was determined.
3
Immunofluorescence Analysis of Inflammasome Proteins
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HaCaT cells or 1° KC were grown on 10 mm2 coverslips at a density of 70’000 cells/coverslip in 24 wells plates. Cells were fixed in 4% paraformaldehyde (PFA) in PBS for 10 min at RT, permeabilized with 0.25% of Triton X-100 in PBS for 10 min and then blocked in PBS containing 1% BSA at room temperature for 30 min as previously described [44 (link)]. Coverslips were incubated with primary antibody ASC (Cat. NBP1-78977 Novus Biological, Littleton, CO, USA) 1:100, NLRP1 (sc-166368 Santa Cruz Biotechnology Inc., Dallas, TX, USA) 1:50, 4HNE (ab46545, Abcam, Cambridge, UK) 1:400, DPP9 (ab42080, Abcam, Cambridge, UK)1:400, UBR2 (18852-1-AP, Proteintech Group Inc., Rosemont, IL, USA) 1: 150 in 0.25% BSA/PBS overnight at 4°C. The Alexa Fluor Fluorochrome-conjugated secondary antibodies (A11004 Alexa Fluor 568, A11008 Alexa Fluor 488 Invitrogen, ThermoFisher Scientific, USA) were used at a dilution of 1:1000 in PBS-BSA 0.25% for 1 h at RT. Nuclei were stained with DAPI (D1306 Invitrogen ThermoFisher Scientific, Waltham, MA, USA) after removal of secondary antibody. Coverslips were mounted onto glass slides using PermaFluor Aqueous Mounting Medium (TA-006-FM, ThermoFisher Scientific, Waltham, MA, USA), and examined using a Zeiss Z1 AxioObserver LSM10 confocal microscope equipped at 40x and 60x magnification. Images were quantified using ImageJ software.
4
Western Blot Analysis of Inflammasome Proteins
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Cells were lysed with RIPA (Radio-Immunoprecipitation Assay) buffer containing protease inhibitors (Calbiochem, USA). 30 μg of total protein was loaded onto a SDSPAGE gel and electroblotted onto Hybond ECL membrane (GE Healthcare, UK). The blots were stained with primary antibodies (AIM2 (1:1000, Cell Signaling Technology, USA), NLRP1 (1:200, Santa Cruz Biotechnology, USA), NLRP2 (1:1000, Proteintech, USA), NLRP3 (1:1000, Adipogen, USA), NAIP (1:1000, Adipogen, USA), ASC (1:1000, Cell Signaling Technology, USA), Caspase 1 (1:200, Santa Cruz Biotechnology, USA), IL1β (1:1000, R & D, USA), Tubulin (!;1000, Cell Signaling Technology, USA), Actin (1:1000, Cell Signaling Technology, USA) overnight at 4°C after blocked with 5% low fat milk (Bio-Rad, USA). The blots were washed with TBST and incubated with a secondary HRP-linked antibody (1:5000, Santa Cruz Biotechnology, USA). Blots were washed TBST before visualization using ECL reagents according to manufacturer’s instructions (Perkin Elmer, USA). Each protein bands’ intensities were normalized against its loading control band intensity for quantification using ImageJ. Briefly, an area of interest rectangle was created around each band and the integrated density was measured, background subtracted, and divided by its specific loading control band.
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