The paraffin sections of 4 μm brain tissue were routinely dewaxed to water, microwave heated with sodium citrate antigen repair solution for 10 min for antigen repair, washed with PBS, and sealed with 10% sheep serum (Solebo, SL038) at room temperature for 1 h. The blocking buffer was vacuumed, and diluted IBA1 primary antibody (1: 400, Wako, 019-19741) and rabbit anti-NLRP3 (PA579740, 1:1,000, Thermo Fisher, USA). Then the sample was incubated overnight at 4°C, and rinsed with 1× PBS for three times prior to the incubation with the second antibodies including Goat anti-rabbit IgG-H&L (Alexa Fluor® 594)(1:1000, Abcam, ab150080) and Goat Anti-Rat IgG H&L (Alexa Fluor® 488) (1:1000, Abcam, ab150165) at room temperature in dark for 1 h. Each sample was incubated with DAPI (Solebo, C0065) at dark for 10 min. After staining, the staining solution was removed and rinsed with 1× PBS for three times. The slides were sealed quenchable sealing tablets and observed under Fluorescence microscope.
Goat anti rabbit igg h l alexa fluor 594
Manufactured by Abcam
Sourced in United Kingdom, United States, Japan
Goat anti-rabbit IgG H&L (Alexa Fluor® 594) is a secondary antibody conjugated with Alexa Fluor® 594 dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg h l alexa fluor 488, by Abcam (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (6 mentions)
Paraformaldehyde (pfa), by Sangon (4 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (3 mentions)
Fluorescence microscope, by Olympus (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Anti fluorescence attenuation mounting tablets, by Solarbio (2 mentions)
Anti nlrp3, by Bioss Antibodies (2 mentions)
Fluorescence microscopy, by Olympus (2 mentions)
Antifading medium, by Solarbio (2 mentions)
12cir 1, by Thermo Fisher Scientific (2 mentions)
Rabbit anti nfκb p65 d14e12 xp, by Cell Signaling Technology (2 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (2 mentions)
30 mm glass bottom dishes, by Matsunami (2 mentions)
X100 5ml, by Merck Group (2 mentions)
Hpa004117 100ul, by Merck Group (2 mentions)
Biorevo bz 9000 fluorescent microscope, by Keyence (2 mentions)
Anti fgfr4, by Santa Cruz Biotechnology (2 mentions)
Anti p stat3, by Cell Signaling Technology (2 mentions)
Anti p src, by Cell Signaling Technology (2 mentions)
35 protocols using goat anti rabbit igg h l alexa fluor 594
1
Immunofluorescence Analysis of NLRP3 and IBA1
2
Collagen II Immunofluorescence of Human Chondrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human chondrocytes with passage 3 were cultured at a density of 3.3 × 104 cells/cm2 in Falcon® 4‐well Chambered cell culture slides. Cells were stained for collagen II by immunofluorescence once they reached ~90% confluence. Chondrocytes were fixed with 10% formalin for 10 min after washing with PBS three times. Fixed cells were incubated for 10 min with PBS containing 0.1% Triton to increase the penetration of the antibody. After washing with PBS three times for 5 min each, cells then were incubated with 10% normal goat serum in PBST (PBS + 0.1% Tween 20) for 30 min to block unspecific binding of the antibodies. Cells were incubated in the diluted anti‐collagen II antibody (1:1,000 dilution, ab34712, Abcam, Cambridge, MA) with 10% normal goat serum in PBST in a humidified chamber overnight at 4°C. Cells were then incubated with goat anti‐rabbit IgG H&L (Alexa Fluor® 594) (1:1,000 dilution, ab150080, Abcam, Cambridge, MA) in 10% normal goat serum in PBST BSA for 1 hr at room temperature in dark. After washing three times with PBS for 5 min each in the dark, the slides were mounted using VECTASHIELD Hardset mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA). Three separate fields per stain were imaged under Nikon's Eclipse E800 fluorescence microscope (Avon, MA). The images were captured at 20× magnification.
3
Antibody and Reagent Procurement for Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit monoclonal anti-IRF3, GAPDH, eGFP-Tag, HA-Tag and HRP-conjugated goat anti-rabbit IgG antibodies, and mouse monoclonal anti-Myc-Tag Flag-Tag antibody were purchased from Cell Signaling Technology (USA). The JetPRIME kit was purchased from Polyplus Transfection (France) and Double-Luciferase Reporter Assay Kit was purchased from TransGen (China). HRP-conjugated goat anti-mouse-IgG antibody, protease inhibitor, and phosphatase inhibitor were provided from CWBIO (China). The Pierce Crosslink Magnetic IP/Co-IP Kit was purchased from Thermo Scientific (USA). Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) were purchased from Abcam (USA). TRIM21 Polyclonal antibody was purchased from Proteintech (USA). MG132, chloroquine diphosphate (CQ), 3-methyladenine (3-MA) and Z-VAD-FMK were purchased from MedChemExpress (MCE) (USA).
4
Apoptosis Induction Visualization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AGS and HGC‐27 cells were plated into Sigma Nunc® Lab‐Tek® II chambered coverglass 8 wells Sigma‐Aldrich at 10,000 cells per chamber in complete medium and incubated for 24 hrs before use. The medium was replaced with ABT‐737, DMC or the combination and then cultured for 4 hrs. Cells were then rinsed with PBS twice before fixation by 4% formaldehyde for 20 min. at room temperature. Cells were then rinsed with PBS for three times and permeabilized by 0.2% Triton X‐100 for 10 min. After washing with PBS, cells were blocked by 5% BSA for 30 min. and incubated with primary antibody overnight at 4°C. Cells were washed by PBS and incubated with secondary antibody (Goat Anti‐Rabbit IgG H&L, Alexa Fluor® 594; Abcam Inc.) for 30 min. The slides were sealed with coverglasses using ProLong® Gold antifade reagent with DAPI (Invitrogen™; Thermofisher Scientific), and immediately observed by confocal microscope (Leica SP8; Leica, Wetzlar, Germany). Apoptosis‐inducing factor was visualized by excitation at 552 nm and its fluorescence emission was observed using a 570–710 nm band‐pass filter. DAPI was excited at 405 nm and the emission was detected ranging from 420 to 540 nm.
5
Immunofluorescence Staining of FUT8 in Ishikawa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
First, we placed autoclaved coverslips on the 24-well plate and the Ishikawa cells were seeded into each well at a density of 1.0 × 105 cells/well. After 24 h, we removed the medium and washed the coverslips with PBS. The slides were fixed with 4% formaldehyde phosphate on ice for 5 min. Then, they were blocked with 4% normal goat serum and 0.2% triton-X at room temperature for 20 min. They were incubated with the anti-FUT8 rabbit polyclonal antibody (Proteintech Group, Inc, IL, USA) at 4 °C overnight. After washing with PBS, they were incubated with the secondary antibody (Goat Anti-Rabbit IgG H&L Alexa Fluor® 594) (Abcam, UK) at room temperature for 1 h. The samples were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, INC., CA, USA) and imaged using an OLYMPUS IX83 inverted microscope (OLYMPUS CO., Japan). The images were taken at 400 × and 1000 × magnification. FUT8 was dyed fluorescent bright red and the nucleus was dyed fluorescent blue.
6
Colocation of RACK1, NLRP3, and NEK7
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the colocalization of RACK1, NLRP3 and NEK7, cells were prepared as described above and transfected with the pcDNA3.1-eGFP-NEK7 plasmid for 48 h. Then, the cells were infected with PmCQ2 for 3 or 4 h. After infection, the cells were washed three times with PBS and fixed in 4% paraformaldehyde (Sango Biotech, Shanghai, China) for 30 min at room temperature (RT). After three wash steps, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and then blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at RT. After three wash steps, the cells were stained with primary antibodies including anti-NLRP3 (Bioss, Beijing, China), anti-ASC (Santa Cruz, CA, USA) and CoraLite®594-conjugated RACK1 (Proteintech, China) at 4 ℃ overnight. Next, secondary Abs including goat anti-mouse IgG (H&L) Alexa Fluor 488 (Abcam, UK), goat anti-rabbit IgG (H&L) Alexa Fluor 594 (Abcam, UK) and ABflo™ 647-conjugated goat anti-rabbit IgG (H&L) (Abclonal, China) were added after washing with PBS and incubated for 1 h at RT. Subsequently, DAPI (Beyotime Biotechnology, Shanghai, China) was added and incubated in the dark for 5 min. Finally, anti-fluorescence attenuation mounting tablets (Solarbio, Beijing, China) were used, and the results were observed using fluorescence microscopy (Olympus, Tokyo, Japan).
7
Immunofluorescent Detection of TXNDC9
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, xylene dewaxed and alcohol-rehydrated paraffin sections were placed in a bottle filled with a 0.01 M trisodium citrate solution and microwaved. After heating, slides were thoroughly rinsed in cool running water for 5 min. Sections were immersed in 3% H2O2 at room temperature for 30 min to block any endogenous peroxidase activity. Add rabbit anti-TXNDC9 (Abcam, Cambridge, UK), diluted 1:200 at an appropriate concentration to the tissue, and incubate at room temperature for 2 h, or in a moist chamber overnight. Adding fluorescently conjugated secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor 594) (Abcam, Cambridge, UK, ab150080), and incubate for 30 min at room temperature in the dark. Add a small volume of mounting media containing DAPI (Abcam, Cambridge, USA, ab104139) stain and add a coverslip of 15 μl is typically enough to cover a tissue underneath a coverslip fully.
8
Neural Stem Cell Differentiation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NSCs were inoculated at a density of 5 × 104 cells/ml into 24-well plates with polylysine-coated glass slides in differentiation solution (DMEM/F-12 supplemented with 1% fetal bovine serum) which was changed after 2 h (Guan et al., 2015 (link)). Bpv(pic) was added to the intervention group at a final concentration of 200 nmol/L, and all cells were cultured for a further 7 days. Cells were then incubated with the following primary antibodies at 4°C for 16 h: rabbit anti-rat βIII tubulin antibody (diluted 1:1000; Abcam), mouse anti-rat glial fibrillary acidic protein (GFAP) antibody (diluted 1:1000; Abcam), and rabbit anti-rat receptor interacting protein (RIP) antibody (diluted 1:1000; Abcam). They were then incubated with goat anti-rabbit IgG H&L (Alexa Fluor® 594) (diluted 1:1000; Abcam) secondary antibodies at 20°C for 2 h. DNA was stained by immediately incubating the slides in 4′,6-diamidino-2-phenylindole (0.2 mg/ml) for 2 min. Slides were stored in the dark at 4°C, then six fields of view per slide were randomly selected. The percentage of positively staining cells in each field was calculated under an Olympus IX71 microscope, and the average value was compared between control and intervention groups.
9
Immunofluorescence Analysis of NLRP3 and ASC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were prepared in 48-well plates and treated with LPS and CATH-2 as described above. After 6 h incubation, cells were washed three times with PBS and fixed in 4% paraformaldehyde (Sango Biotech, Shanghai, China) for 20 min at room temperature (RT). After three wash steps, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. Subsequently, cells were blocked with 5% Bovine Serum Albumin (BSA) in PBS for 30 min. Then, cells were stained with primary antibody containing anti-NLRP3 (Bioss, Beijing, China) and anti-ASC (Santa cruz, CA, USA) for 1 h at RT. After the wash steps, cells were incubated with Goat anti-mouse IgG (H&L) Alexa fluor 488 and Goat anti-rabbit IgG (H&L) Alexa fluor 594 (Abcam, UK) for 1 h. DAPI (Beyotime Biotechnology, Shanghai, China) was added for 5 min to visualize cell nuclei. Finally, cells were washed and maintained in antifading medium (Solarbio, Beijing, China). Cells were observed using the fluorescence microscopy (Olympus, Tokyo, Japan).
10
Immunofluorescence Staining of Tubulin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AGS and HGC-27 cells were plated incomplete medium in SigmaNunc®Lab-Tek®IIChamber Slide systems (8 chamber; Sigma-Aldrich, St Louis, MO, USA) at 10 000 cells per chamber and incubated for 24 h before use. The medium was replaced with DI (12 or 30 μmol/L) or paclitaxel (0.5 μmol/L)11 (link) and cultured for 24 h. The cells were then rinsed with PBS twice before fixation by 4% formaldehyde for 20 min at room temperature. The cells were rinsed with PBS three times and permeabilized with 0.2% Triton X-100 for 10 min. After washing with PBS, the cells were blocked with 5% BSA for 30 min and incubated overnight with the primary antibody at 4 °C. The cells were washed with PBS and incubated with the secondary antibody (Goat Anti-Rabbit IgG H&L, Alexa Fluor® 594, Abcam, Cambridge, MA, USA) for 30 min. The slides were sealed with cover glasses using the ProLong® Gold anti-fade reagent with DAPI (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA) and immediately observed by confocal microscopy (Leica SP8, Mannheim, Germany). Tubulin was visualized at 552 nm excitation, and fluorescence emission was observed using a 570–710 nm bandpass filter. DAPI was excited at 405 nm, and emission was detected from 420 to 540 nm.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!