Spinal cord sections were incubated in a blocking solution containing 0.1 M PBS, 0.3% Triton X-100 (PBST), and 3% BSA (Sigma Aldrich) for 1 h at room temperature. Spinal cord transverse cryosections were incubated overnight at room temperature with a rabbit anti-Iba1 (1:500, Wako Chemicals) and a rat anti-α5 integrin (1:400, BioLegend) in PBST. Anti-Iba1 antibody was revealed with a goat anti-rabbit Alexa Fluor-594 secondary antibody (Life Technologies), anti-α5 integrin antibody was revealed with a goat anti-rat Alexa Fluor-488 secondary antibody (Life Technologies) and sections were stained for DAPI before mounting the slides. Sciatic nerves were incubated in a blocking solution containing 0.1 M PBS, 0.3% Triton X-100 (PBST), and 3% BSA (Sigma Aldrich) for 1 h at room temperature. Sciatic nerves were sequentially stained first with a rat anti-α5 integrin antibody (1:400, BioLegend) overnight at room temperature and revealed with a goat anti-rat Alexa Fluor-488 secondary antibody (Life Technologies). On the second day, sciatic nerves were incubated with a cocktail of rat anti-CD11b (1:400, BD Biosciences), rat anti-CD68 (1:400, AbD Serotec), and rat anti-F4/80 (1:100, AbD Serotec) antibodies overnight at room temperature and revealed with a goat anti-rat Alexa Fluor-594 secondary antibody (Life Technologies).
Alexa fluor 488 goat anti rat secondary antibody
Manufactured by Thermo Fisher Scientific
The Alexa Fluor 488 goat anti-rat secondary antibody is a fluorescently labeled reagent used to detect and visualize rat primary antibodies in various immunological techniques, such as immunofluorescence, flow cytometry, and Western blotting. The Alexa Fluor 488 dye provides bright and photostable green fluorescence, enabling sensitive detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti cd68, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Rat anti f4 80, by Bio-Rad (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Alexa fluor 594 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rat anti cd11b, by BD (1 mentions)
12 well plate, by BD (1 mentions)
Alexa fluor 594 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Eclipse ti fluorescence microscope, by Nikon (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
4 protocols using alexa fluor 488 goat anti rat secondary antibody
1
Spinal Cord and Sciatic Nerve Immunostaining
2
Quantifying Tumor-Infiltrating CD8+ Cells
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Tumors were frozen in Tissue-Tek OCT and stored at -80°C. Frozen tumors were then sectioned (8 μm), fixed in 75%/25% acetone/methanol for 5 minutes, and washed using PBS. Slides were incubated with normal goat serum (10% in PBS) for 1 hour at room temperature, washed, and incubated with rat anti-mouse CD8 (AbDSerotec) at 1:100 dilution in 10% goat serum/PBS overnight at 4°C. After washing, Alexa Fluor 488 goat anti-rat secondary antibody (Invitrogen) was added to the slides (1:1000) for one hour at room temperature. Prolong Gold antifade with 4´, 6-diamidino-2-phenylindole (DAPI) mounting media (Invitrogen) was added after an additional wash and cover slips were attached. Positive cells and DAPI stained nuclei were counted in three random high-powered microscopic fields per slide and expressed as a mean. The number of positive cells in the field was expressed as # of CD8+ cells per mm2 tumor area. Data are shown as the mean and SEM for 3 mice/group.
3
Immunofluorescence Staining of Integrin Subunits
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IF was performed as described previously (32 (link)). Briefly, 5×104 T84 cells were seeded onto serum-pretreated coverslips in a 12-well plate (Falcon), allowed to adhere for 48h under normal culture conditions and processed for IF experiments. For α6 and β4 staining, cells were fixed in 2% paraformaldehyde, non-specific sites were blocked for 1 h at room temperature with 5% Blotto–PBS (pH 7.4) and both primary and secondary antibodies were diluted in 5% Blotto–PBS (pH 7.4). For α6A and α6B staining, cells were fixed in MeOH and EtOH, respectively. Non-specific sites were blocked for 1 h at room temperature in a 2% bovine serum albumin solution in PBS (pH 7.4) and both primary and secondary antibodies were diluted in 2% bovine serum albumin–PBS (pH 7.4). Cells were treated with a 0.2% Triton X-100 solution for 5 min prior to antibody incubation. Primary antibodies were detected with Alexa Fluor 488 or 594 goat anti-mouse secondary antibodies (Invitrogen, A11017, A11032) and Alexa Fluor 488 goat anti-rat secondary antibody (Invitrogen, A11006).
4
Immunofluorescent Staining of Tumor Sections
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Frozen tumor sections were fixed with cold acetone, acetone plus chloroform (1:1), and acetone. Tissue sections were blocked with blocking buffer (5% normal horse serum and 1% normal goat serum in PBS) and incubated with rat anti-mouse CD8α (clone YTS105.18, AbD Serotec, Raleigh, NC), rat anti-mouse CD4 (clone RM4-5, BD Pharmingen, San Jose, CA), or rat anti-mouse NKp46 antibody (clone 29A1.4, Biolegend, San Diego, CA) overnight at 4°C. The next day, tissue sections were blocked and incubated with goat anti-rat Alexa fluor 488 secondary antibody (Life Technologies, Grand Island, NY) for 1 hour at room temperature. Tissues were then blocked and incubated with second primary antibody NKG2D-biotin antibody (1:50; R&D Systems, Minneapolis, MN) overnight at 4°C and second secondary antibody streptavidin-conjugated Alexa fluor 594 (Life Technologies, Grand Island, NY) for 1 hour at room temperature. Rat IgG was used as the negative control. Nuclei were counterstained with Hoechst 33258 (1:10,000) (Life Technologies, Grand Island, NY). Tumor sections were mounted in antifade fluorescence mounting medium (Life Technologies, Grand Island, NY). Slides were visualized under the Nikon eclipse Ti fluorescence microscope (Nikon, Melville, NY) with use of appropriate filters (original magnification, 100×).
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