Cryosections were stored at −20C° freezers until use. Sections were postfixed in 4% paraformaldehyde for 15 minutes, blocked with 2% BSA/TBST for 30 minutes and incubated with rat anti-CD31 monoclonal antibody (1:100, AbD Serotec MCA2388), overnight at 4°C, and subsequently with Alexa Fluor 633-conjugated goat anti-rat IgG (1:400, Invitrogen A21087) for 3 hours at 4°C. For lipid staining, cryosections were gently rinsed with TBS and incubated with LipidTOX Deep Red (1:200, Invitrogen H34477) for 30 minutes at room temperature. Sections were further incubated with DAPI (4',6-diamidino-2-phenylindole, Invitrogen D1306) to stain nuclei. Stained samples were cover-slipped and mounted in a mounting medium for fluorescence imaging (Vectashield H-1000, Vector Labs). The edge of the cover slip was coated with commercially available transparent nail polish.
Alexa fluor 633 conjugated goat anti rat igg
Manufactured by Thermo Fisher Scientific
Alexa Fluor 633-conjugated goat anti-rat IgG is a secondary antibody conjugated with Alexa Fluor 633 dye. It is designed to detect and visualize rat immunoglobulin G (IgG) in various immunoassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
Vectashield h 1000, by Vector Laboratories (2 mentions)
Lipidtox deep red, by Thermo Fisher Scientific (2 mentions)
Rat anti cd31 monoclonal antibody, by Bio-Rad (2 mentions)
Sm568p, by OriGene (2 mentions)
Ck548, by Merck Group (1 mentions)
Interleukin 4 (il 4), by GenScript (1 mentions)
Ck636, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
5 protocols using alexa fluor 633 conjugated goat anti rat igg
1
Immunohistochemistry of Vascular Endothelium
2
Immunohistochemistry of Vascular Endothelium
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Cryosections were stored at −20C° freezers until use. Sections were postfixed in 4% paraformaldehyde for 15 minutes, blocked with 2% BSA/TBST for 30 minutes and incubated with rat anti-CD31 monoclonal antibody (1:100, AbD Serotec MCA2388), overnight at 4°C, and subsequently with Alexa Fluor 633-conjugated goat anti-rat IgG (1:400, Invitrogen A21087) for 3 hours at 4°C. For lipid staining, cryosections were gently rinsed with TBS and incubated with LipidTOX Deep Red (1:200, Invitrogen H34477) for 30 minutes at room temperature. Sections were further incubated with DAPI (4',6-diamidino-2-phenylindole, Invitrogen D1306) to stain nuclei. Stained samples were cover-slipped and mounted in a mounting medium for fluorescence imaging (Vectashield H-1000, Vector Labs). The edge of the cover slip was coated with commercially available transparent nail polish.
3
Antibody Characterization for Cell Signaling
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The rat mAb M1/70, which recognizes the mouse αM integrin subunit, was purified from the conditioned media of hybridoma cells (obtained from The American Tissue Culture Collection, Manassas, VA) using protein A agarose. The mouse anti-talin (T3287) and anti-vinculin (V9131) mAbs and rabbit anti-I/S-afadin polyclonal antibody (A0224) were from Sigma (St. Louis, MO).
The rabbit anti-paxillin (32084) and anti-JAM-A (180821) polyclonal antibodies and rabbit anticortactin mAb conjugated to Alexa Fluor 555 were from Abcam (Cambridge, MA). The rat antinectin-2 (502-57) mAb was from Santa Cruz Biotechnology (Dallas, TX). The mouse anti-Ecadherin mAb (24E10) and rabbit anti-Myosin IIa polyclonal antibody (34035) were from Cell Signaling (Danvers, MA). The rat anti-SIRPα/CD172a polyclonal antibody (P84; 552371) was from BD Bioscience (San Jose, CA). The rabbit anti-MT1-MMP-14 polyclonal antibody (14552-1-AP) was from Proteintech (Rosemont, IL). The rabbit anti-ZO-1 polyclonal antibody (61-7300) and secondary antibodies Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 633conjugated goat anti-rat IgG were from Invitrogen (Carlsbad, CA). Vibrant DiD membranelabeling reagent was from Thermo Fisher (Waltham, MA). Brewer's thioglycollate (TG), wiskostatin, and the Arp2/3 inhibitors CK-548 and CK-636 were from Sigma (St. Louis, MO). IL-4 was from Genscript (Piscataway, NJ).
The rabbit anti-paxillin (32084) and anti-JAM-A (180821) polyclonal antibodies and rabbit anticortactin mAb conjugated to Alexa Fluor 555 were from Abcam (Cambridge, MA). The rat antinectin-2 (502-57) mAb was from Santa Cruz Biotechnology (Dallas, TX). The mouse anti-Ecadherin mAb (24E10) and rabbit anti-Myosin IIa polyclonal antibody (34035) were from Cell Signaling (Danvers, MA). The rat anti-SIRPα/CD172a polyclonal antibody (P84; 552371) was from BD Bioscience (San Jose, CA). The rabbit anti-MT1-MMP-14 polyclonal antibody (14552-1-AP) was from Proteintech (Rosemont, IL). The rabbit anti-ZO-1 polyclonal antibody (61-7300) and secondary antibodies Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 633conjugated goat anti-rat IgG were from Invitrogen (Carlsbad, CA). Vibrant DiD membranelabeling reagent was from Thermo Fisher (Waltham, MA). Brewer's thioglycollate (TG), wiskostatin, and the Arp2/3 inhibitors CK-548 and CK-636 were from Sigma (St. Louis, MO). IL-4 was from Genscript (Piscataway, NJ).
4
Immunofluorescence Analysis of Mitochondrial Proteins
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Human dermal fibroblasts were subjected to γ-irradiation as described above and seeded in 6-well plates on coverslips. Alternatively, cells were transfected with miR-15b inhibitors in the absence or presence of siRNA duplexes against SIRT4 as described below. Cells were fixed four days later in 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 20 min followed by a blocking step with 4% BSA/0.05% saponin for 30 min at room temperature. Subsequently, cells were costained with primary antibodies against the mitochondrial marker MTCO2 (abcam, ab3298; 1:500), SIRT4 (Santa Cruz Biotechnology, Inc., sc-135053; 1:200), and α-Tubulin (Acris antibodies, SM568P, 1:500) overnight at 4°C. Secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG, Alexa Fluor 543-conjugated goat anti-rabbit IgG, and Alexa Fluor 633-conjugated goat anti-rat IgG) were from Life Technologies and used at a dilution of 1:500 for 1 hr at room temperature. Analyses were performed with a LSM510-Meta confocal microscope (Zeiss) equipped with 40/1.3 or 63/1.4 immersion objectives and excitation wavelengths of 488 nm, 543 nm, and 633 nm. Cells were examined and pictures were taken at the Z-stack level (0.5 μm) and when indicated further analysed using the ImageJ software v1.49k as described in the suppl. Materials & Methods part.
5
Quantification of Parkin Recruitment to Mitochondria
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HEK293 cells stably expressing eGFP, SIRT4-eGFP, SIRT4(H161Y)-eGFP, or SIRT4(Δ28N)-eGFP were transfected with pCMV6-mCherry-Parkin (PARK2) and treated with 10 µM CCCP together with 100 nM Bafilomycin A1 for two hours on the next day, as described above. Cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 20 min followed by a blocking step with 4% BSA/0.05% saponin for 30 min at room temperature. Cells were co-stained with primary antibodies against the mitochondrial marker MTC02 (abcam, ab3298; 1:500), and α-Tubulin/TUBA1B (Acris antibodies, SM568P; 1:500) overnight at 4°C. Secondary antibodies (Alexa Fluor 546-conjugated goat anti-mouse IgG and Alexa Fluor 633-conjugated goat anti-rat IgG) were from Life Technologies and used at a dilution of 1:500 for one hour at room temperature. Analyzes were performed with a LSM510-Meta confocal microscope (Zeiss) equipped with 40/1.3 immersion objectives and emission wavelengths of 468 nm, 488 nm, 543 nm, and 633 nm. Quantification of mCherry-Parkin dots was performed based on the mitochondrial content (MTC02 signal) using ImageJ software v1.49k and a specific macro (Suppl. Material & Methods ).
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