Immediately following the completion of a stimulation protocol, 16% paraformaldehyde (PFA) was added to each well to a final concentration of 4%, and cells were incubated in PFA in the dark for 10 min. Cells were then permeabilized with 100/50 μL (for 96/384-well plates) with phosphate buffered saline (PBS) + 0.1% Triton-X for 10 min. Cells were then further permeabilized with ice cold methanol for 10 min. After permeabilization, cells were blocked with 1% BSA at room temperature for 30 min. Primary antibody was diluted in PBS + 1% BSA according to the manufacturer's recommendation for immunofluorescence (phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Cell Signaling #4370, 1:400 dilution; phospho-Akt (Ser473), Cell Signaling Technologies #9271, 1:800 dilution). 96/384-well wells were incubated with 50/25 μL of antibody dilution for 2 hr at room temperature (RT). Samples were incubated at room temperature in primary antibody (for two hours, after which primary antibody was removed and samples underwent five washes in PBS + 0.1% TWEEN-20 (PBS-T). Cells were then incubated with secondary antibody (Jackson Immunoresearch Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG (H+L)) and DAPI (ThermoFisher, #D1306, 300 nM) in PBS-T + 0.1% BSA for 1 hour at RT. Secondary antibody was removed, samples underwent 5 washes with PBS-T. Samples were imaged in PBS-T.
Alexa fluor 488 affinipure goat anti rabbit igg h l
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG (H+L) is a secondary antibody conjugated with Alexa Fluor® 488 fluorescent dye. It is designed to detect and visualize primary antibodies raised in rabbit.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (2 mentions)
Phospho akt ser473, by Cell Signaling Technology (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti satb2, by Abcam (1 mentions)
Paraformaldehyde aqueous solution, by Electron Microscopy Sciences (1 mentions)
Ab14196, by Abcam (1 mentions)
Ab2413, by Abcam (1 mentions)
Mab4470, by R&D Systems (1 mentions)
Tritc phalloidin, by Yeasen (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
Hoechst, by Beyotime (1 mentions)
6 protocols using alexa fluor 488 affinipure goat anti rabbit igg h l
1
Immunofluorescence Staining of Phosphorylated Proteins
2
Incisor Apical Papilla Characterization
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The use and care of C57BL/6 mice and Sprague‐Dawley (SD) rats in this study followed an animal protocol approved by the Research Ethics Committee of College of Stomatology, Chongqing Medical University, Chongqing, China (CQHS‐REC‐2020（LSNo.55)）. Mandibular specimens from C57BL/6 mice (4‐week‐old males and females) were extracted for isolation of the incisor apical papilla. Immunofluorescence and haematoxylin and eosin (H&E) staining were carried out. Anti‐Satb2 (1:600, Abcam, Cambridge, UK) or anti‐Bmp9 (1:100; ThermoFisher Scientific, Waltham, USA) antibody and Alexa Fluor® 488‐AffiniPure goat anti‐rabbit IgG (H + L) were used as the primary and secondary antibodies, respectively. Control IgGs and an absence of primary antibody were used as negative controls.
3
Immunocytochemistry for Phosphorylated ERK1/2
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Immediately following completion of a stimulation protocol,16% paraformaldehyde (Paraformaldehyde Aqueous Solution, Electron Microscopy Sciences, catalog number 15710) was added to each well to a final concentration of 4%, and cells were incubated for 10 min in the dark. Cells were then permeabilized 1X PBS + 0.1% Triton X-100 for 10 min at room temperature (RT). Cells were further permeabilized with ice-cold 100% methanol at −20 °C for 10 min. After permeabilization, the cells were blocked with 1% BSA in 1X PBS for 30 min at RT. The cells were then incubated in primary antibody diluted in 1X PBS + 0.1% BSA (phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Cell Signaling, catalog number 4370L, 1:400 dilution) at 4 °C overnight. After overnight incubation, the primary antibody was removed and the plate was washed 5 times in PBS + 0.1% Tween-20 (PBS-T). Cells were then incubated with secondary antibody (Jackson Immunoresearch Alexa Fluor 488 AffiniPure goat anti-rabbit IgG (H+L), 1:500) and DAPI (ThermoFisher Scientific, catalog number D1306, 300 nM) in 1X PBS + 0.1% BSA for 1 h at RT. The secondary antibody was removed, and the plate was washed 5 times in PBS-T.
4
Histological Analysis of mdx Mouse Muscle
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After the euthanized mice were skinned, the gastrocnemius muscles and diaphragms were carefully removed for fixation in 4% paraformaldehyde for 24 h. Then, the muscles were embedded in paraffin, sectioned to 4 μm, and stained with Sirius Red to observe muscle fibrosis and hematoxylin and eosin to observe muscle histopathology and central nuclei as features of mdx mouse muscle. Muscle restoration was detected by incubating fixed gastrocnemius sections with myosin heavy chain antibody (#MAB4470, at 1/1000 dilution; R&D Systems, MN, USA), annexin V antibody (ab14196, at 1/500 dilution, Abcam), and fibronectin antibody (ab2413, at 1/200 dilution, Abcam) for 18 h at 4 °C, then with Alexa Fluor® 488 AffiniPure Goat Anti-mouse IgG (H+L) (A10680, Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor® 488 AffiniPure Goat Anti-rabbit IgG (H+L) (A27034, Thermo Fisher Scientific), and Alexa Fluor® 594 AffiniPure Goat Anti-rabbit IgG (H+L) (A11037, Thermo Fisher Scientific) secondary antibodies. Sections were counterstained with Hoechst 33342 (H1339, Thermo Fisher Scientific).
5
Immunofluorescence Staining of BmVasa in Spermatocytes
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The anti-BmVasa antibody BmVasa-R1 was described previously [55 (link),67 (link)]. Immunofluorescence staining experiments using BmVasa-R1 were performed using spermatocytes and sperm bundles isolated from excised testes from the fifth instar larvae stage to adult stage animals. The collected sperm were fixed for 1 h. After two washes with PBS, samples were incubated in the primary antibody-blocking solution mixture overnight at 4°C (PBS containing 0.1% Triton X-100 and 1% bovine serum albumin). After two washes with PBS, samples were incubated with the secondary antibody, TRITC Phalloidin (YEASEN, China), and Hoechst (Beyotime, China) for 1 h at room temperature. Samples were washed three times with PBS and subsequently mounted in the antifade medium (YEASEN, China). Images were taken on an Olympus BX53 microscope (Japan). Antibodies and dilutions used were as follows: BmVasa-R1, 1:200; Alexa Fluor 488 AffiniPure Goat Anti-Rabbit IgG (H+L) (Thermo Fisher, USA), 1:500; TRITC Phalloidin (YEASEN, China), 1:500.
6
Immunofluorescence Staining of Phosphorylated Proteins
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