Sections were deparaffinized and rehydrated in xylene followed by decreasing alcohol gradients. Antigen retrieval was performed in citrate buffer pH 6 for 20 minutes in the microwave. Blocking was performed with 10% serum in 0.5% Tween-20 PBS for 2 hours at room temperature. Primary antibody (GFP; Santa Cruz sc-9996) staining was performed overnight at 4°C at a concentration of 1:100 in 1% serum 0.5% Tween-20 PBS. Secondary antibody (Alexa Flour 488 goat anti-mouse; Invitrogen #A11001) staining was performed for 2 hours at roomperature at a concentration of 1:250 in 1% serum 0.5% Tween-20 PBS. Finally, sections were stained with DAPi for 20 minutes at room temperature prior to mounting with Flouromount-G (Southern Biotech). All washes were performed in 0.5% Tween-20 PBS. Sections stained following the protocol above with the exception of no primary antibody were used as negative controls. Metastatic lesions were visualized, counted, and imaged using a Leica DM5500 B upright automated microscope.
Alexa flour 488 goat anti mouse
Manufactured by Thermo Fisher Scientific
Sourced in United States, Denmark
Alexa Fluor 488 goat anti-mouse is a fluorescently-labeled secondary antibody that binds to mouse primary antibodies. It is used in immunofluorescence and other applications that require the detection of mouse-derived proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 900 confocal microscope, by Zeiss (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Flouromount g, by Southern Biotech (2 mentions)
Sc 9996, by Santa Cruz Biotechnology (2 mentions)
Dm5500 b upright automated microscope, by Leica (2 mentions)
Sheep anti digoxigenin, by Roche (2 mentions)
Alexa fluor 555 donkey anti sheep, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (2 mentions)
Mouse anti biotin primary antibodies, by Thermo Fisher Scientific (2 mentions)
Western blocking reagent, by Roche (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Alexa flour 594 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Evos fl digital inverted fluorescence microscope, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab2723, by Abcam (1 mentions)
Ab5392, by Abcam (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Alexa fluor 633 goat anti chicken, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
4 6 diamino 2 phenylindole dapi, by Merck Group (1 mentions)
15 protocols using alexa flour 488 goat anti mouse
1
Immunohistochemistry Protocol for GFP Detection
2
Hippocampal Neuron Immunofluorescence Imaging
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Immunofluorescence was performed as previously reported (Ismail et al. 2017 (link)) on hippocampal primary neurons after administration of the treatments indicated above. Single and double immunofluorescence was performed with the following primary antibodies: chicken anti-MAP2 (1:10 000 dilution, Abcam, ab5392) and mouse anti-PSD95 (1:1000 dilution, Abcam, ab2723). Cells were then incubated for 1 h at room temperature at a dilution of 1:1000 with secondary antibodies; Alexa fluor 633 goat antichicken and Alexa flour 488 goat antimouse (Invitrogen, A11035 and A11055, respectively). Identification of the nuclei of cell bodies was performed using 4, 6 diamino-2-phenylindole (DAPI; Sigma) and the F-actin was stained by Alexa Fluor 488-Phalloidin (1:1000; Life Technologies). Omission of the primary antibody was done as a control staining. Coverslips were finally mounted onto glass slides using fluorescence mounting medium (DAKO Cytomation, Glostrup, Denmark). Imaging was performed with a Zeiss (LSM 510 META) confocal laser scanning system using a 488 nm Argon laser, 633 nm Helium–neon laser and UV 405 nm. The fluorescence of DAPI, Alexa 488 and Alexa 633 was recorded through separate channels.
3
Bag-1 and Beclin 1 Colocalization Assay
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Cells were seeded as 1 × 105 cells per well in 12-well plate containing poly-L-lysine coated coverslips, and co-transfected with His6Bag-1S or His6Bag-1L and His6Beclin 1 plasmid. 48 h post-transfection, culture medium was discarded, and cells were washed with phosphate buffered saline (PBS) solution. Cells were fixed in pre-chilled 4% paraformaldehyde and incubated for 10 min at room temperature, then, washed three times with PBS. Cells were blocked by 1 h incubation in blocking solution (3% BSA, 0.1% Triton-X 100 in PBS) and were incubated with appropriate primary antibodies overnight at 4 °C. Primary antibodies used were mouse anti-Bag-1 (1:200), rabbit anti-Beclin 1 (1:200). Following washing, cells were incubated for 1 h at RT with the secondary antibody (Alexa Flour® 488 goat anti-mouse or Alexa Flour® 594 goat anti-rabbit; 1:300 for both, Invitrogen, Carlsbad, CA, USA). After extensive washing, coverslips were mounted on slides using ProLong Diamond Antifade mounting medium containing DAPI (P36962, Thermo Fisher, Waltheim, MA, USA). Confocal images were obtained via a Leica TCS SP2 SE confocal microscope (Leica, Buffalo Grove, IL, USA).
4
Immunohistochemistry Protocol for GFP Detection
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Sections were deparaffinized and rehydrated in xylene followed by decreasing alcohol gradients. Antigen retrieval was performed in citrate buffer pH 6 for 20 minutes in the microwave. Blocking was performed with 10% serum in 0.5% Tween-20 PBS for 2 hours at room temperature. Primary antibody (GFP; Santa Cruz sc-9996) staining was performed overnight at 4°C at a concentration of 1:100 in 1% serum 0.5% Tween-20 PBS. Secondary antibody (Alexa Flour 488 goat anti-mouse; Invitrogen #A11001) staining was performed for 2 hours at roomperature at a concentration of 1:250 in 1% serum 0.5% Tween-20 PBS. Finally, sections were stained with DAPi for 20 minutes at room temperature prior to mounting with Flouromount-G (Southern Biotech). All washes were performed in 0.5% Tween-20 PBS. Sections stained following the protocol above with the exception of no primary antibody were used as negative controls. Metastatic lesions were visualized, counted, and imaged using a Leica DM5500 B upright automated microscope.
5
Immunofluorescence Staining of MEF2C and Myosin
6
Whole-Mount in situ Hybridization of Drosophila Embryos
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Embryos were dechorionated and fixed in fixation buffer (1 ml of 5× PBS, 4 ml of 37% formaldehyde and 5 ml of Heptane) for ∼25 min at room temperature. Vitelline membrane was then removed by shaking embryos in a biphasic mixture of heptane and methanol for ∼1 min. Antisense RNA probes labeled with digoxigenin and biotin were used. Hybridization was performed at 55°C for overnight in hybridization buffer (50% formamide, 5× SSC, 50 μg/ml Heparin, 100 μg/ml salmon sperm DNA, 0.1% Tween-20). Subsequently, embryos were washed with hybridization buffer at 55°C and incubated with Western Blocking Reagent (Roche) at room temperature for 1 h. Then, embryos were incubated with sheep anti-digoxigenin (Roche) and mouse anti-biotin primary antibodies (Invitrogen) at 4°C for overnight, followed by incubation with Alexa Fluor 555 donkey anti-sheep (Invitrogen) and Alexa Flour 488 goat anti-mouse (Invitrogen) fluorescent secondary antibodies at room temperature for 1 h. DNA was stained with DAPI, and embryos were mounted in ProLong Gold Antifade Mountant (Thermo Fisher). Imaging was performed on a Zeiss LSM 900 confocal microscope. Plan-Apochromat 20×/0.8 N.A. objective was used. Images were captured in 16-bit. Maximum projections were obtained for all z-sections, and resulting images were shown. Brightness of images was linearly adjusted using Fiji (https://fiji.sc ).
7
Whole-Mount In Situ Hybridization in Embryos
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Embryos were dechorionated and fixed in fixation buffer (1 ml of 5x PBS, 4 ml of 37% formaldehyde and 5 ml of Heptane) for ~25 min at room temperature. Vitelline membrane was then removed by shaking embryos in a biphasic mixture of heptane and methanol for ~1 min. Antisense RNA probes labeled with digoxigenin and biotin were used.
Hybridization was performed at 55 C overnight in hybridization buffer (50% formamide, 5x SSC, 50 μg/ml Heparin, 100 μg/ml salmon sperm DNA, 0.1% Tween-20).
Subsequently, embryos were washed with hybridization buffer at 55 C and incubated with Western Blocking Reagent (Roche) at room temperature for one hour. Then, embryos were incubated with sheep anti-digoxigenin (Roche) and mouse anti-biotin primary antibodies (Invitrogen) at 4 C for overnight, followed by incubation with Alexa Fluor 555 donkey anti-sheep (Invitrogen) and Alexa Flour 488 goat anti-mouse (Invitrogen) fluorescent secondary antibodies at room temperature for one hour. DNA was stained with DAPI, and embryos were mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Imaging was performed on a Zeiss LSM 900 confocal microscope.
Plan-Apochromat 20x / 0.8 N.A. objective was used. Images were captured in 16-bit.
Maximum projections were obtained for all z-sections, and resulting images were shown.
Brightness of images was linearly adjusted using Fiji (https://fiji.sc).
Hybridization was performed at 55 C overnight in hybridization buffer (50% formamide, 5x SSC, 50 μg/ml Heparin, 100 μg/ml salmon sperm DNA, 0.1% Tween-20).
Subsequently, embryos were washed with hybridization buffer at 55 C and incubated with Western Blocking Reagent (Roche) at room temperature for one hour. Then, embryos were incubated with sheep anti-digoxigenin (Roche) and mouse anti-biotin primary antibodies (Invitrogen) at 4 C for overnight, followed by incubation with Alexa Fluor 555 donkey anti-sheep (Invitrogen) and Alexa Flour 488 goat anti-mouse (Invitrogen) fluorescent secondary antibodies at room temperature for one hour. DNA was stained with DAPI, and embryos were mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Imaging was performed on a Zeiss LSM 900 confocal microscope.
Plan-Apochromat 20x / 0.8 N.A. objective was used. Images were captured in 16-bit.
Maximum projections were obtained for all z-sections, and resulting images were shown.
Brightness of images was linearly adjusted using Fiji (https://fiji.sc).
8
Immunocytochemical analysis of myoblast purity
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Immunocytochemistry was used to determine myoblast cell purity. Skeletal muscle myoblast cells were seeded at a density of 5 × 103 cells per well in a 96-well plate. After cells were washed with phosphate buffer saline (PBS), the cells were fixed in cold 100% ethanol for 10 minutes followed by incubation with 1% FBS for 30 minutes, with 3x PBS washes in between these procedures. Then, the cells were incubated sequentially with an anti-desmin monoclonal antibody in a dark environment for 1 h (D33, Dako, Glostrup, Denmark) and Alexa Flour 488 goat anti-mouse in a dark environment for 1 h 45 min (Life Technologies, Carlsbad, CA, USA), with 3x PBS washes in between incubations. Hoechst 33342 (Life Technologies, Carlsbad, CA, USA) was then used to visualize the cell nuclei. The cells were viewed under an EVOS FL Digital Inverted Fluorescence Microscope (Life Technologies, Carlsbad, CA, USA). The percentage of desmin-positive cells was determined by examining a minimum of 50 cells from three independent cultures [24 (link)].
9
Immunofluorescence Analysis of Myoblast Differentiation
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Immunofluorescence staining on myoblasts in culture was performed using an antibody specific for desmin, at a dilution of 1:50 (D33; DAKO, Glostrup, Denmark). Skeletal myoblast cells were washed with phosphate-buffered saline (PBS) and fixed in 100% cold ethanol, followed by incubation in 1% fetal bovine serum (FBS). The skeletal myoblast cells were washed again with PBS and incubated consequently with anti-desmin monoclonal antibody (D33, DAKO, Glostrup, Denmark) and Alexa Flour 488 goat anti-mouse in dark environment (Life Technologies, Carlsbad, CA, USA) with PBS washing in between the two antibodies incubation. Cell nuclei were visualized by Hoechst 33342 (Life Technologies, Carlsbad, CA, USA). The desmin staining was viewed under EVOS FL Digital Inverted Fluorescence Microscope (Life Technologies, Carlsbad, CA, USA). The morphological changes of the myoblast cell were observed throughout the differentiation days.
10
CD31 Immunofluorescence in Hydrogels
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After fixation and washing with PBS the samples were permeabilized using 0.1% Triton X-100 for 10 min. The samples were blocked with 2% BSA and 2% goat serum in PBS for 3 h and incubated over night with the primary antibodies mouse anti-CD31 (BD 555444; 1:100) at 4°C. After 3 washes with PBS/0.1% BSA, the secondary antibody Alexa Flour 488 goat anti-mouse (Life Technologies 1:200) was added and incubated over night at 4°C. Samples were washed and incubated with Hoechst 33342 (Life Technologies; 1:200) over night at 4°C. Fluorescent images were captured using a Dragonfly Spinning Disk confocal microscope (Andor). At least three images were taken at different positions within each hydrogel. Fields and samples for imaging and quantification were chosen randomly for all experiments.
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