Following PBS washing for 3 × 5 min, the retinal sections were incubated with 0.1% Triton for 10 min, subjected to normal rabbit serum (Invitrogen, CA, USA) to block non-specific binding sites, and then incubated at 37 °C for 30 min to discard serum. Then, primary antibody against Vimentin (NB300-223, 1:5000) or CHX10 (NBP1-84476, 1:1000) (Novus, St. Louis, MO, USA) was incubated with sections overnight in a humidified box at 4 °C (the NC group was treated with 0.01 mmol/L of PBS). Then, sections were washed thrice with PBS for 5 min, followed by incubation with the secondary antibody labeled with FITC fluorescence (ab6662, 1:1000, Abcam, USA) or Texas Red (ab6787, 1:1000, Abcam, USA) in a humidified box at 37 °C for 40 min. Following 5 min of PBS rinsing thrice, sections were sealed with water-soluble resin. Pictures were captured with a fluorescence microscope (Olympus IX71, Tokyo, Japan). Analyses of images were performed by using Fiji [16 (link)] and associated plugins as previously described [17 (link)].
Ab6787
Manufactured by Abcam
Sourced in United States
Ab6787 is a primary antibody for detecting a specific target protein in samples. It is designed for use in immunoassay applications, such as Western blotting, immunohistochemistry, and ELISA. The antibody is purified and validated for its specificity and sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (2 mentions)
Supersignal west femto maximum, by Thermo Fisher Scientific (2 mentions)
Normal rabbit serum, by Thermo Fisher Scientific (1 mentions)
Ab6662, by Abcam (1 mentions)
Nb300 223, by Novus Biologicals (1 mentions)
Nbp1 84476, by Novus Biologicals (1 mentions)
β actin, by Merck Group (1 mentions)
Ab7260, by Abcam (1 mentions)
Ab104224, by Abcam (1 mentions)
Ab10062, by Abcam (1 mentions)
Pa5 78290, by Thermo Fisher Scientific (1 mentions)
Ab6717, by Abcam (1 mentions)
6 protocols using ab6787
1
Immunofluorescence Staining of Retinal Sections
2
Western Blot Analysis of Exosome Markers
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EVs were lysed using 50 μL of RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], 1 mM NaF) and incubated with agitation for 30 min at 4 °C followed by sonication for 30 s. Samples were loaded with 6× Laemmli buffer (5:1, 60 µg/well) onto 10% SDS-PAGE and then transferred on nitrocellulose membrane. The membrane was blocked with 5% milk in TBS-T (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20) and stained with primary antibodies to exosome markers (EXOAB-KIT-1 for CD63, CD9, CD81 and Hsp70, SBI) 1:1000 or to β-actin (A1978, Sigma) 1:5000 in 5% milk in TBS-T overnight at 4 °C. Membranes were washed 3× with TBS-T and incubated for 1 h with goat anti-rabbit HRP-conjugated antibodies (Ab6721, Abcam, Cambridge, MA, USA) or with goat anti-mouse HRP-conjugated antibodies (Ab6787; Abcam) diluted 1:5000 in 5% milk in TBS-T. Membranes were washed 3 times with TBS-T and the chemiluminescent signal was developed with SuperSignal West Femto Maximum (Thermo Fisher Scientific, Oxford, UK) and detected with an X-ray film with 2 h exposure.
3
Immunofluorescent Quantification of GFAP
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For immunofluorescence staining, the cells were fixed with 4% PFA solution for 20 min and permeabilized using 0.1% Triton-X-100 for 5 min. Then, cells were blocked using 3% BSA and incubated with anti-GFAP (ab7260, Abcam) antibody for one hour. After PBS wash, cells were incubated with FITC-labeled secondary antibody (ab6787, Abcam) for 60 min in RT. For nuclear counterstaining, the slides were exposed to 1 µg/ml 4′,6-diamidino-2-phenylindole (DAPI), and stained cells were visualized using an invert fluorescence microscopy. The percentage of GFAP+ was calculated according to the following formula GFAP+ + DAPI+ cells/DAPI+ cells × 100. The assessment was performed three times.
4
Exosome Marker Detection by Western Blot
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EV/EMNV preparations or HEK-293T cells were lysed using 50 μL of RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0,1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS], 1 mM NaF) and incubated with agitation for 30 min at 4 °C followed by sonication for 30 seconds. Samples were loaded with 6× Laemmli buffer (5:1, 60 µg/well) onto 10% SDS-PAAG and then transferred on nitrocellulose membrane. Membrane was blocked with 5% milk in TBS-T (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20) and stained with primary antibodies to exosome markers (EXOAB-KIT-1 for CD63, CD9, CD81 and Hsp70, SBI) 1:1000 or to β-actin (A1978, Sigma) 1:5000 in 5% milk in TBS-T overnight at 4 °C. Membranes were washed 3× with TBS-T and incubated for 1 h with goat anti-rabbit HRP-conjugated antibodies (Ab6721, Abcam, Cambridge, MA, USA) or with goat anti-mouse HRP-conjugated antibodies (Ab6787; Abcam) diluted 1:5000 in 5% milk in TBS-T. Membranes were washed 3× with TBS-T and chemiluminescent signal was developed with SuperSignal West Femto Maximum (Thermo-Fisher) and detected with an X-ray film with 2 h exposure.
5
GLUT5 Antibody Staining Protocol
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The GLUT5 (Slc2a5) primary antibody (sc-271055) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and the secondary antibody (ab6787) was obtained from Abcam (Cambridge, MA, USA). The primary antibody was used with incubation buffer at a 1:200 dilution, while the secondary antibody was used at a dilution of 1:1000, as recommended. All the cells were fixed in 4% paraformaldehyde and blocked for 1 h at room temperature. Incubation for primary and secondary antibodies was 2 h and 1 h, respectively, at room temperature. A PBS rinse was carried out between each step. Imaging was carried out immediately.
6
Immunofluorescence Staining of Brain Sections
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Immuno uorescence was performed as described in our previously published articles 25, 26 . The brain sections were incubated in a primary antibody solution containing rabbit anti-ROCK2 antibody (1:700; #PA5-78290, Invitrogen) and mouse anti-NeuN antibody (1:1000; #ab104224, Abcam) or mouse anti-GFAP antibody (1:1000; #ab10062, Abcam) that was dissolved in the rinse buffer for 12 h at 4 °C. Before secondary antibody incubation, the sections were washed 3 times for 30 min in PBS. The sections were then incubated in a secondary antibody solution containing FITC-conjugated goat anti-rabbit IgG (1:2000; #ab6717, Abcam) and Texas Red-conjugated goat anti-mouse IgG (1:2000; #ab6787, Abcam) in the rinse buffer for 4 h at 4 °C. After washing 3 times for 30 min in PBS, the sections were coverslipped with anti-fading medium.
Images of double immuno uorescence staining were obtained using a confocal laser-scanning microscope system (Nikon C2+, Tokyo, Japan).
Images were analyzed with ImageJ software (ver. 1.46; NIH, USA).The ratios of cell number of single-positive cells and double-positive cells (%)
was evaluated as described in the article 27 .
Images of double immuno uorescence staining were obtained using a confocal laser-scanning microscope system (Nikon C2+, Tokyo, Japan).
Images were analyzed with ImageJ software (ver. 1.46; NIH, USA).The ratios of cell number of single-positive cells and double-positive cells (%)
was evaluated as described in the article 27 .
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