Cells were plated onto an eight-well Lab-Tek chambered coverglass (Thermo Fisher Scientific) and cultured to around 70%. Before imaging, the medium was discarded, and the cells were washed with PBS twice. Then, eGFP-bFGF protein solution (50 nM and 500 nM) was applied to the cells for 5 min at RT. After washed three times with PBS, cells fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 5 min at room temperature (RT). The resulting cells were placed in blocking buffer (3% BSA in PBS) for 1 h followed by washing, and labelled with Anti-Rab4 Antibody- Early Endosome Marker (abcam, Britain, 1:170 dilution) for overnight at 4 °C. After three washing steps with PBS, the cells were incubated with Cy3-labelled Goat Anti-Rabbit IgG(H + L) (Beyotime Biotechnology, China, 1:500 dilution) antibody for 2 h at RT. The immune stained cells were examined under a confocal laser scanning microscopy using an inverted Leica SP8 microscope, equipped with lasers for 405-nm, 488-nm, 552-nm excitation. Images were acquired using a 100×objective and LAS X software 3.2.
Cy3 labelled goat anti rabbit igg h l
Manufactured by Beyotime
Sourced in China
Cy3-labelled Goat Anti-Rabbit IgG(H + L) is a secondary antibody conjugated with the fluorescent dye Cy3. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and techniques.
Automatically generated - may contain errors
Lab products found in correlation
Sp8 microscope, by Leica (1 mentions)
Lab tek chambered coverglass, by Thermo Fisher Scientific (1 mentions)
Sodium citrate antigen retrieval solution, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Donepezil hydrochloride, by Maclin Biochemical Technology (1 mentions)
Malondialdehyde mda elisa kit, by Nanjing Jiancheng (1 mentions)
Rabbit anti gapdh, by Bioworld Technology (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Triglyceride assay kit, by Nanjing Jiancheng (1 mentions)
Confocal microscopy, by Zeiss (1 mentions)
Alexa fluor 488 labelled goat anti mouse igg h l, by Beyotime (1 mentions)
Alexa fluor 647 labeled goat anti mouse igg h l, by Beyotime (1 mentions)
Alexa fluor 488 labelled goat anti rabbit igg h l, by Beyotime (1 mentions)
Cy3 labelled goat anti mouse igg h l, by Beyotime (1 mentions)
5 protocols using cy3 labelled goat anti rabbit igg h l
1
Visualizing eGFP-bFGF Endocytosis in Cells
2
Immunofluorescent Staining of TH Neurons
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All sections were incubated under standard conditions as follows: (a) in 4% paraformaldehyde (Beyotime Biotech Inc, Shanghai, China) for 5–10 min at room temperature; (b) in sodium citrate antigen retrieval solution (Beyotime Biotech Inc, Shanghai, China) treated under microwave at low power heat for 10 min; (c) in seal solution (Beyotime Biotech Inc, Shanghai, China) for 1 h at room temperature or 24 h at 4 °C; (d) TH-polyclonal antibody (1:500; Shanghai Youke Biotechnology Co. Ltd., Shanghai, China) for 24 h at 4 °C; and (e) CY3-labelled goat anti-rabbit IgG H + L (1:100; Beyotime Biotech Inc, Shanghai, China) for 1–2 h at room temperature. The sections were washed three times with PBS after each incubation step for 5 min each. After the sections were dehydrated, the DAPI-containing anti-fluorescence quencher was added dropwise. The sections were incubated with a cover glass in the dark for 10 min. Nail polish was dropped at the edge of the coverslip to prevent slippage, and the sections were imaged with a fluorescence microscope (BX63; Olympus Corporation, Tokyo, Japan).
3
JUND Protein Immunofluorescence in PBMCs
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PBMCs were seeded at a density of 1 × 105 cells per well in 24-well culture plates which were precoated with Poly-D-lysine (Sigma, P6407). The cells were fixed with 4% paraformaldehyde for 0.5 h and blocked with 0.4% Triton X-100 and 5% goat serum for an hour. Then the cells were incubated with JUND antibody (CST, 5000) at 4°C overnight. Following a meticulous washing, the cells were incubated with secondary antibody (Cy3-labelled goat anti-rabbit IgG (H + L), Beyotime, A0516) for 1 h at 37°C and the images were captured by microscopy (Leica, Germany).
4
Herbal Neuroprotection Protocol
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The 6 raw herbs of DSS were purchased from Kangmei Pharmaceutical Limited Company (Guangzhou, China). Donepezil hydrochloride (D849374) and scopolamine hydrobromide trihydrate (S860151) were from Macklin (Shanghai, China); FITC-dextran (#46944) was from Sigma system (ChemiDoc MP, Bio-Rad, California, USA); total cholesterol Assay Kit (A111-1-1) and Triglyceride Assay Kit (A110-1-1) were from Nanjing Jiancheng; and Malondialdehyde (MDA) ELISA Kit (JL13339), Adiponectin (ADPN) Kit (JL20696D), Low-Density Lipoprotein Cholesterol (LDL-C) Kit (JL20313), High-Density Lipoprotein Cholesterol (HDL-C) Kit (JL20356), Tumour Necrosis Factor-α Kit (TNF-α) (JL10484) and Interleukin 6 (IL-6) Kit (JL20313) were from Jiang Lai Biological (Shanghai, China). Antibodies included rabbit anti-ZO-1 (PB9234, Boster, Pleasanton, CA, USA), rabbit anti-OCLN (A01246-2, Boster), rabbit anti-OCLN (#91131, CST, Danvers, MA, USA) in brain immunofluorescence, rabbit anti-ZO-1 (#13663, CST) in brain immunofluorescence, rabbit anti-PPAR-γ (#2435, CST), Cy3-labelled Goat Anti-Rabbit IgG (H+L) (A0516, Beyotime, Shanghai, China), rabbit anti-LXR alpha+beta (ab21669, Abcam, Cambridge, MA, USA), rabbit anti-actin (#2118, CST), and rabbit anti-GAPDH (AP0063, Bioworld, St. Louis Park, MN, USA).
5
Immunofluorescent Analysis of Retinal Microglia
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Eyes obtained on day P17 were placed into 4% paraformaldehyde for 2 h. Then the retinas were sheared into flat mounts and blocked with 5% goat serum and 0.4% Triton X-100 for 1 h, followed by incubation with primary antibodies at 4 °C overnight. Then the retinas were washed carefully and incubated with secondary antibody combinations for 1 h. Images were taken by confocal microscopy (Zeiss, Germany). CellSens Dimension software was used to determine the number of microglia (Iba1+). Relative fluorescence intensity was measured by ImageJ. The following primary antibodies were used: CD31 (Abcam, ab9498, diluted 1:1000); IBA1 (WAKO, 019–19,741, diluted 1:1000); Pan-Kla (PTM-1401, diluted 1:200);YY1-K183la (PTM, diluted 1:200); YY1 (Proteintech, 66281–1-lg, diluted 1:200); p300 (Santa Cruz, sc48343, diluted 1:200); and Ki67 (Abcam, ab16667, diluted 1:200). The following secondary antibodies were used: Alexa Fluor 488-labelled goat anti-rabbit IgG (H + L) (Beyotime, A0423); Alexa Fluor 488-labelled goat anti-mouse IgG (H + L) (Beyotime, A0428); Cy3-labelled donkey anti-goat IgG (H + L) (Beyotime, A0502); Cy3-labelled goat anti-mouse IgG (H + L) (Beyotime, A0521); Cy3-labelled goat anti-rabbit IgG (H + L) (Beyotime, A0516); Alexa Fluor 647-labeled goat anti-mouse IgG (H + L) (Beyotime, A0473). All secondary antibodies were diluted 1:500.
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