Myofibroblast cells were cultured in a μ-Slide 8 well (ibidi, 80826) to become fully confluent (90% to 100%). Cells were fixed with paraformaldehyde for 10 min at room temperature and then permeabilized with 0.1% Triton X-100 in PBS. Nonspecific staining was blocked with Background Sniper (Biocare Medical, BS966). Cells were stained by incubation with the monoclonal anti-α-SMA antibody (Sigma, A2547) diluted 1:200 and using Alexa Fluor 488 donkey anti-mouse IgG secondary antibody (ThermoFisher, A-21202) to detect fluorescence. The cells were washed, excess liquid was aspirated, and secondary antibody solution was added (1 to 10 μg/ml) (Alexa Fluor 488 donkey anti-mouse IgG secondary antibody; ThermoFisher, A-21202). 4′,6-Diamidino-2-phenylindole (DAPI) staining was used for nuclei. Confocal images were obtained with a Nikon Eclipse Ti2 (×60 lens objective; Nikon, Canada) and quantified by the methodology of Dössel et al. (36 ). Fluorescence intensity measurements were obtained from entire cells and analyzed with Image J software. Control specimens were identical to experimental specimens, except they were exposed to an irrelevant isotype-matched antibody.
Alexa fluor 488 donkey anti mouse igg secondary antibody
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488 donkey anti-mouse IgG secondary antibody is a fluorescent-labeled secondary antibody designed for use in immunoassays and other applications that require the detection of mouse primary antibodies. The Alexa Fluor 488 dye provides a bright green fluorescent signal when excited at the appropriate wavelength.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade mountant, by Thermo Fisher Scientific (2 mentions)
Tsa plus cyanine 5, by PerkinElmer (2 mentions)
Alexa fluor 594 goat anti chicken igy secondary antibody, by Thermo Fisher Scientific (2 mentions)
Ab205402, by Abcam (2 mentions)
Ab1218, by Abcam (2 mentions)
μ slide 8 well, by Ibidi (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
Background sniper, by Biocare Medical (1 mentions)
Monoclonal anti αsma antibody, by Merck Group (1 mentions)
Ab4648, by Abcam (1 mentions)
Ab34735, by Abcam (1 mentions)
Cytopainter phalloidin ifluor 488 reagent, by Abcam (1 mentions)
Ix51 microscope, by Olympus (1 mentions)
Cm1850 cryostat, by Leica (1 mentions)
Tcs sp5 confocal laser scanning microscope, by Leica (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Mouse anti neurofilament 2h3, by Developmental Studies Hybridoma Bank (1 mentions)
Alexa fluor 555 α bungarotoxin α btx, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
6 protocols using alexa fluor 488 donkey anti mouse igg secondary antibody
1
Immunofluorescence Analysis of Myofibroblasts
2
Multiplexed smFISH and Immunocytochemistry
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smFISH targeting nascent RNA of HES1 or BCL11A were performed using RNAscope® Multiplex Fluorescent Reagent Kit v2(ACDBio). Probes binding the intronic region of target genes were designed and synthesized by ACDBio. FISH signal was labeled with TSA Plus Cyanine 5 (Perkin Elmer). Immunocytochemistry was carried out after FISH procedure [22 ]. Antibodies targeting GFP(Abcam, ab1218), mCherry (Abcam, ab205402), GFAP (Ab4648) and SATB2 (Abcam, ab34735) were incubated overnight. Secondary antibodies including Alexa Fluor 594 Goat anti-chicken IgY secondary antibody (Thermo Fisher Scientific, A11042), Alexa Fluor 488 donkey anti-mouse IgG secondary antibody (Thermo Fisher Scientific, A21202), Alexa Fluor 546 donkey anti-mouse IgG secondary antibody (Thermo Fisher Scientific, A10036) and Alexa Fluor 488 donkey anti-rabbit IgG secondary antibody (Thermo Fisher Scientific, A21206) were incubated at RT for 1hr. Nuclei are stained with DAPI for 5 min before mounting with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific, P36930).
3
Nascent HES1 RNA in Cortical Neurons
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Primary cells dissected from developmental dorsal cortex were cultured on coverslips and infected with lenti-virus expressing either GFP or mCherry. Cells were fixed in 4% PFA on Day 4 for staining. RNAscope and Immunocytochemistry staining smFISH targeting nascent RNA of HES1 was performed using RNAscope® Multiplex Fluorescent Reagent Kit v2(ACDBio). Probes binding the intronic region of HES1 were designed and synthesized by ACDBio. FISH signal was labeled with TSA Plus Cyanine 5 (Perkin Elmer). Immunocytochemistry was carried out after FISH procedure. Antibodies targeting GFP(Abcam, ab1218) and mCherry (Abcam, ab205402) were incubated overnight. Secondary antibodies including Alexa Fluor 594 Goat anti-chicken IgY secondary antibody (Thermo Fisher Scientific, A11042) and Alexa Fluor 488 donkey anti-mouse IgG secondary antibody (Thermo Fisher Scientific, A21202) were incubated at RT for 1hr. Nuclei are stained with DAPI for 5 min before mounting with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific, P36930).
4
Quantitative Analysis of Osteoclast Fusion
5
Localization of VP28 protein in shrimp tissues
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Samples were fixed overnight with 4% paraformaldehyde, dehydrated with 20% sucrose for 24 h, and 30% sucrose for 24 h, sequentially. For juvenile L. vannamei, the cephalothorax, and abdomen (about 0.5 cm in length) were collected from each animal. For subadult L. vannamei, a section of midgut about 0.5 cm in length was collected from each animal. The samples were placed in an optimal cutting temperature compound, transferred to liquid nitrogen, and stored at −80 °C prior to sectioning. The samples were cross-sectioned into 5-μm thick slices using a Leica CM1850 cryostat. The slices were dried in an oven overnight and sequentially fixed with cold acetone for 10 min, washed with PBS, probed with the anti-VP28 antibody, and incubated with the Alexa Fluor 488 donkey anti-mouse IgG secondary antibody (Life Technologies). The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Tissue sections were observed with Confocal Laser Scanning Microscope Tcs SP5 (Leica), and fluorescence microscope DM6000B (Leica).
6
Neuromuscular Junction Staining Protocol
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TVA, ETA, FDB, and lumbrical muscles of both the hands and feet were dissected (Murray et al., 2014; Sleigh et al., 2014a; Tarpey et al., 2018) and stained as previously described (Sleigh et al., 2017 (Sleigh et al., , 2014b)) . Briefly, muscles were excised and fixed for 10 min in 4% (w/v) paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA) in phosphate buffered saline (PBS), before being permeabilised for 30 min in 2% (v/v) Triton X-100 in PBS and blocked for 30 min in 4% (w/v) bovine serum albumin and 1% (v/v) Triton X-100 in PBS. Muscles were then probed overnight at 4˚C in blocking solution with 1/250 mouse anti-neurofilament (2H3, Developmental Studies Hybridoma Bank [DSHB], Iowa City, IA, supernatant) and 1/25 mouse pan anti-synaptic vesicle 2 (SV2, DSHB, supernatant) antibodies to co-visualise axons and presynaptic nerve terminals, respectively. The following day muscles were washed several times with PBS before being incubated with 1/1,000 Alexa Fluor 488 donkey anti-mouse IgG secondary antibody (Life Technologies, Carlsbad, CA, A-21202) and 1/1,000 Alexa Fluor 555 α-bungarotoxin (α-BTX, Life Technologies, B35451) in PBS for 2 h. Muscles were washed with PBS and then mounted in Fluoromount-G (Southern Biotech, Birmingham, AL, 0100-01) on microscope slides for imaging. All five muscles from each mouse were processed in parallel.
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