Prior to immunofluorescence staining, the KGNs in 6‑well plates were rinsed three times with PBS and then fixed with 4% paraformaldehyde for 15 min at room temperature. After rinsed three times with PBS, cells were blocked with goat serum albumin (C0265, Beyotime Biotechnology) for 60 min at room temperature. Then, incubate cells at 4˚C overnight in a solution containing LC3 antibody. Subsequently after PBST rinsing, the cells were stained with an Alexa Fluor 488‑conjugated anti‑mouse antibody (SA00006-1, Proteintech Technology) for 60 min at 25˚C. Finally, rinsed with PBST and incubated with DAPI for 5 min in dark. The images were captured by a fluorescence microscope (Olympus Corporation, Tokyo, Japan).
Goat serum albumin
Manufactured by Beyotime
Sourced in China
Goat serum albumin is a protein derived from the serum of goats. It is commonly used in various laboratory applications as a standard, stabilizer, or blocking agent.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (3 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (3 mentions)
Alexa fluor 594 and 488 conjugated secondary antibodies, by Proteintech (2 mentions)
Fv10i, by Olympus (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
C fos, by Abcam (1 mentions)
Rpe65, by Novus Biologicals (1 mentions)
C rel, by Abcam (1 mentions)
Tecnai g2 spirit twin, by Thermo Fisher Scientific (1 mentions)
Fish kit, by RiboBio (1 mentions)
Gb21303, by Wuhan Servicebio Technology (1 mentions)
Confocal microscopy, by Leica (1 mentions)
Gb25301, by Wuhan Servicebio Technology (1 mentions)
Gsh and gssg assay kit, by Beyotime (1 mentions)
Rat alexafluor488, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Lsm 880 multiphoton confocal microscope, by Zeiss (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
12 protocols using goat serum albumin
1
Immunofluorescence Staining of KGNs
2
Visualizing PGC-1α Expression in Cardiomyocytes
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NRCMs were washed with PBS and fixed with 4% paraformaldehyde for 15 min at 4°C. Following a further wash, cells were blocked with a 1% goat serum albumin (Beyotime Institute of Biotechnology, Haimen, China) for 1 h and permeabilized in PBS with 0.1% Triton X-100 for 5 min at room temperature. Anti-PGC-1α-N-terminal antibody (1:100; cat. no. ab191838; Abcam) was applied overnight at 4°C. Fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (1:200; cat. no. sc-2012; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was subsequently added for 2 h at room temperature. For the visualization of nuclei, cells fixed at 4°C were incubated with DAPI for 10 min at room temperature. In addition, live cells were infected with mCherry-NT-PGC-1α or mCherry adenovirus and incubated with Hoechst 33258 for 10 min. Cells were observed under a confocal microscope (Olympus FV10i' magnifications, ×10 or 60).
3
Immunofluorescence Analysis of Neural Markers
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Eyes on slides were permeated with 0.1% Triton X‐100 in PBS at room temperature for 20 min. Then, the slides were blocked (5% goat serum albumin (Beyotime)) for 1 h at room temperature, and tissues were incubated with primary antibodies against GFAP (Abcam, 1:1000), GS (Abcam, 1:1000), Tuj1 (Abcam, 1:1000), pS6 (Abcam, 1:1000), and c‐fos (Abcam, 1:1000) at 4°C overnight. The slides were infiltrated in PBS for 30 min, followed by an hour of incubation with secondary antibodies (Alexa Fluor 488 or Alexa Fluor 568 conjugated, Invitrogen, USA). DAPI was used to mark the nuclei. All slides were shot and analyzed with a Leica TCS 170 SP8 confocal laser scanning microscope (Leica TCS NT, Wetzlar, Germany).
4
Immunofluorescence Analysis of Choroidal Flatmounts
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Immunofluorescence assays were performed on choroidal flat mounts. Briefly, after being fixed, the samples were blocked with 0.3% Triton X-100 and 5% goat serum albumin (Beyotime, Shanghai, China) in PBS for 1 h at room temperature. The tissues were immunostained with primary antibodies against F4/80 (1:500; CST, Beverly, MA, USA), Ym-1 (1:500; Stem Cell Technology, Vancouver, Canada) and isolectin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C, washed three times with PBS, and then stained for 45 min at 37 °C with Alexa Fluor 594- and 488-conjugated secondary antibodies (1:1000; Proteintech, Chicago, IL, USA). The nuclei were stained with 4,6-diamidino-2-phenylindole. Images were visualized using a fluorescence microscope (Olympus, Center Valley, PA, USA) and a Leica TCS SP8 confocal laser scanning microscope (Leica TCS NT, Wetzlar, Germany) [20 (link)].
5
Immunofluorescence Staining Protocol
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Cells were fixed and subsequently blocked for 30 min using 5% goat serum albumin (Beyotime Biotechnology, China). The tissue sections were incubated overnight at 4 °C with specific primary antibodies (as listed in Table 1 ) and with fluorescent secondary antibodies for 1 h at room temperature. Fluorescence images were captured using a fluorescence microscope (Nikon, Tokyo, Japan).
6
Immunofluorescence Analysis of Retinal and RPE Samples
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Mice were killed and perfused intracardially with 4% paraformaldehyde in phosphate-buffered saline (PBS). The eyeballs were enucleated, retinal flatmounts were prepared, RPE-choroid complexes were flatmounted, and cryosections (10 μm) were prepared for immunofluorescence analysis. The samples were immunostained with antibodies against GFP, α-SMA, PDGFRβ, BEST1, CD31, and GS, respectively. The sizes of fibrotic lesions were determined using ImageJ, which measured the areas of GFP-positive lesions or PDGFRβ-positive and α-SMA-positive lesions on RPE-choroid complexes. Cells on cover slips were washed with 1× PBS and fixed in 4% paraformaldehyde in PBS for 20 min. After being fixed, the samples were blocked with 1× PBS containing 0.3% Triton X-100 and 5% goat serum albumin (Beyotime) for 1 h. Then, the cells were incubated with the corresponding antibodies at 4 °C overnight. The antibody information is listed in Supplementary Table 3 .
7
Immunocytochemistry Analysis of Retinal Cells
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Immunocytochemistry assays were performed on retinal sections or in 24-well slide chambers. Briefly, after fixation, the samples were blocked with 0.3% Triton X-100 and 5% goat serum albumin (Beyotime) in PBS for 1 h at room temperature. Tissue sections were immunostained with primary antibodies against RelA (1:300, Cell Signaling Technology), RelB (1:300, Abcam), c-Rel (1:300, Abcam) and RPE65 (1:300, Novus Biologicals, Littleton, CO, USA) overnight at 4 °C. The samples were washed with PBS three times and then were stained for 45 min at 37 °C with Alexa Fluor 594- and 488-conjugated secondary antibodies (1:1 000, Proteintech). The nuclei were marked by 4′,6-diamidino-2-phenylindole. RPE cells were visualized using a fluorescence microscope (Olympus) and a Leica TCS SP8 confocal laser scanning microscope (Leica TCS NT, Wetzlar, Germany).
For ultrastructural analysis, the samples were send to Shanghai FuDan University School of Medicine after fixation in 2.5% glutaraldehyde (dissolved in PBS) at 4 °C. The ultrathin sections were observed under an electron microscope (Tecnai G2 spirit twin, FEI, Eindhoven, The Netherlands).
For ultrastructural analysis, the samples were send to Shanghai FuDan University School of Medicine after fixation in 2.5% glutaraldehyde (dissolved in PBS) at 4 °C. The ultrathin sections were observed under an electron microscope (Tecnai G2 spirit twin, FEI, Eindhoven, The Netherlands).
8
RNA Localization and Protein Expression Analysis in Cells
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Used the FISH Kit (RiboBio, Guangzhou, China) to perform fluorescence in situ hybridization of RNA. Inoculated cells onto a 24-well chamber slide (Ibidi from Martinsried, Germany). Designed and synthesized specific FISH probes targeting UCA1. Next day, removed DMEM and washed the cells. Fixed the cells with 4% PFA. Blocked the cells with 5% goat serum albumin (Beyotime Biotechnology, China). Treated the cells with Triton X-100 (0.5%) at room temperature for 15 min.Washed the cells. Incubated the slide with denaturing probes or negative controls targeting UCA1, U6, and 18S rRNA at 37 °C overnight. Stained the nucleus with DAPI. Obtained the images by a confocal laser scanning microscope (Examiner. Z1, Carl Zeiss, Germany).
Incubated the slides with IGF2BP3 and c-MYC primary antibodies (Table S2) overnight at 4 ℃. Incubated the slides with fluorescent secondary antibodies at room temperature for 1 h. Obtained the images by a confocal laser scanning microscopy.
Incubated the slides with IGF2BP3 and c-MYC primary antibodies (Table S2) overnight at 4 ℃. Incubated the slides with fluorescent secondary antibodies at room temperature for 1 h. Obtained the images by a confocal laser scanning microscopy.
9
Intracellular Glutathione Regulation in BV2 Cells
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BV2 cells were seeded at a density of 2 × 10 5 cells/well in 12-well plates and incubated overnight prior to treatment with LPS, PF-06447475, Erastin, and Ferrostatin-1. To detect intracellular GSH levels, the GSH and GSSG Assay Kit (S0053; Beyotime, Jiangsu, China) was used according to the manufacturer's instructions. Brie y, after treatment, the cells were lysed, and the supernatant was used for GSH detection at a wavelength of 405 nm using a microplate reader (Waltham, MA, United States). Through standard curve to calculate samples glutathione, speci cally, use the formula GSH = total glutathione -GSSG x 2 of GSH content in the samples.
Immuno uorescence BV2 cells were cultured in 12-well plates, xed with PBS containing 0.03% Triton X-100 and 5% goat serum albumin (C0265; Beyotime, Jiangsu, China), and subsequently incubated with primary antibodies against LRRK2 (1:100), p-p65 (1:1000), and GPX4 (1:200) overnight at 4°C. Cy3-conjugated goat antirabbit IgG (GB21303, 1:200; Servicebio) or goat anti-mouse IgG conjugated with Alexa Fluor 488 (GB25301, 1:200; Servicebio) were applied for 2 hour at room temperature. Confocal microscopy (Leica, USA) was used to capture immunostaining images.
Immuno uorescence BV2 cells were cultured in 12-well plates, xed with PBS containing 0.03% Triton X-100 and 5% goat serum albumin (C0265; Beyotime, Jiangsu, China), and subsequently incubated with primary antibodies against LRRK2 (1:100), p-p65 (1:1000), and GPX4 (1:200) overnight at 4°C. Cy3-conjugated goat antirabbit IgG (GB21303, 1:200; Servicebio) or goat anti-mouse IgG conjugated with Alexa Fluor 488 (GB25301, 1:200; Servicebio) were applied for 2 hour at room temperature. Confocal microscopy (Leica, USA) was used to capture immunostaining images.
10
Immunofluorescence Staining of HA-Tagged Cells
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Cells were fixed with 4% paraformaldehyde for 30 min and blocked with PBS containing 5% goat serum albumin (Beyotime) and 0.05% Triton for 1 h. Then, the cells were incubated at 4 °C overnight with antibodies against HA(1:1000, 11,867,431,001, Roche). After being washed with PBS 3 times for 5 min, the cells were incubated with secondary antibody (Rat, Alexa Fluor 488, Invitrogen, USA) for 1 h. Finally, the cells were visualized with a Leica TCS SP8 confocal laser scanning microscope (Leica TCS NT, Wetzlar, Germany) [9 (link)].
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