Total cells on the slides went through permeabilization in 0.3% Triton X-100 after the fixation in 4% paraformaldehyde. After that, goat serum was utilized for blocking. Cells or tissues were later cultivated overnight with anti-GFAP (Abcam, Cambridge, UK). Then, the slide was subjected to 1-h incubation with the Fluorescein (PE)-conjugated Affinipure Goat Anti-Mouse IgG(H+L) (Proteintech, Wuhan, China) at room temperature. Again, cells or tissues were cultivated overnight with anti-beta-Galactosidase (Proteintech, Wuhan, China) or P16INK4a (Abcam, Cambridge, UK) at 4 °C and then subjected to 1 h incubation with the FITC-conjugated Goat Anti-Rabbit IgG(H+L) (Proteintech, Wuhan, China). The counterstaining of the nucleus was accomplished by using DAPI. For immunohistochemistry (IHC), slides were incubated in 0.9% H2O2 for 30 min. Afterwards, the slides were placed in blocking buffer (goat serum 1:20 in PBST/BSA) for 30 min at room temperature. Then, anti-iba1 (Abcam Cambridge, UK) antibody was used, and tissues were further blocked with Biotin for 10 min each. Antibodies were detected using a rabbit peroxidase ABC Kit (MXB Biotechnologies, Guangzhou, China). Each sample was viewed with BX53 Fluorescence microscope (Olympus, Tokyo, Japan).
Fitc conjugated goat anti rabbit igg h l
Manufactured by Proteintech
Sourced in United States
FITC-conjugated Goat Anti-Rabbit IgG(H+L) is a secondary antibody used for detection in immunoassays. It is conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate) and binds to the heavy and light chains of rabbit immunoglobulin G (IgG) antibodies.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti gfap, by Abcam (1 mentions)
Anti iba1, by Abcam (1 mentions)
Bx53 fluorescence microscope, by Olympus (1 mentions)
P16ink4a, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Proteintech (1 mentions)
Rabbit anti hmgb1 monoclonal antibody, by Abcam (1 mentions)
Tcs sp5 2, by Leica (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
3 protocols using fitc conjugated goat anti rabbit igg h l
1
Multiparametric Immunofluorescence Staining Protocol
2
Immunofluorescence Assay for HMGB1 Localization
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Immunofluorescence assays (IFA) for HMGB1 localization and translocation in ANA1 and mouse PMΦs were performed as described previously (Wang et al., 2014 (link)). Briefly, cells cultured on coverslips and subjected to different treatments were fixed, permeabilized, and blocked prior to incubation with the rabbit anti-HMGB1 monoclonal antibody (ABcam, UK, diluted 1:200 in 1% BSA-PBS) for 1 hr at 37°C. The coverslips were washed and then incubated for 1 h at 37°C with FITC-conjugated goat anti-rabbit IgG (H+L) diluted to 1:50 in PBS with 1% BSA (Proteintech, USA). Nuclei were stained with Hoechst 33258 (Sigma–Aldrich, USA) and coverslips were subsequently mounted onto slides. Fluorescence images were obtained through the Leica microsystem (Leica TCS SP5 II, Germany) and epifluorescence optics were obtained using an oil immersion lens with 63× magnification. Collected images and MIFs (mean index of fluorescence) were processed and analyzed using the LAS AF lite 2.2.0 software (Leica). Mouse anti-TgHMGB1a antibodies were used to trace the T. gondii (Wang et al., 2014 (link)).
3
Multiplex Immunofluorescence Analysis of Tumor Immune Cells
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Tumor tissues were fixed in 4% paraformaldehyde and immuno-fluorescence was performed on 5 µm thick paraffin sections after heat-induced antigen retrieval. The following primary antibodies were used: CD4 (1:200, Abcam, Cambridge, UK, ab34276), CD8 (1:200, Abcam, ab101500), CD19 (1:200, CST, #90176s), CD56 (1:200, Abcam, ab21336), CD68 (1:200, Abcam, ab53444), CD11b (1:200, Abcam, ab133357), LY6G (1:200, Abcam, ab25377), and EpCam (1:200, Abcam, ab213500). The HER-2 immunohistochemical reagent was the ready-to-use 4B5 rabbit anti-human monoclonal antibody, purchased from Roche. Subsequently, we incubated the slides with fluorescent secondary antibodies (fluorescein (FITC)-conjugated goat anti-rabbit IgG (H+L), 1:1000, Proteintech, San Diego, CA, USA, SA00003-2; rhodamine (TRITC)-conjugated goat anti-rat IgG (H+L), 1:1000, Proteintech, SA00007-7; Alexa Fluor ® 647-conjugated goat anti-mouse IgG (H+L), 1:1000, Cell Signaling Technology, Danvers, MA, USA, #4410), DAPI (Thermo Scientific, Waltham, MA, USA, #62247), and the Opal 6-Color Manual IHC Kit (Akoya Biosciences, Marlborough, MA, USA, NEL811001KT) for multilabel immunofluorescence analysis. Positive cells were determined and quantified by Perkin Elmer (Waltham, MA, USA) in FormTM system.
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