Apoptosis was measured in mice with MHV-1 pneumonia using a TUNEL immunofluorescence assay. Lungs were harvested from control and MHV-1 infected mice, formalin-fixed, paraffin-embedded, sectioned and then dual stained with Click-iT® Plus TUNEL (Thermofisher) and 4′,6-diamidino-2-phenylindole (DAPI). To confirm MHV-1 infection, the same sections were blocked with 3% BSA in PBS at 37 °C for 1 hour and an MHV A59 Nsp9 antibody (NBP-21671, Novus Biologicals, Centennial, CO, USA) was applied (1:500 for 1 hour at 37 °C). Finally an Alexa Fluor 546 goat anti-mouse secondary antibody (Thermofisher, A11035) was applied at 1:400 and the slides were incubated for 1 hour at 37 °C. Images were then captured on a Leica SP8 confocal microscope using a 63X 1.4 NA objective and a white light laser at 488 nm. Emission acquisition gating was 510–530 nm (for TUNEL). A 405 nm laser diode was used for DAPI excitation and the emission acquisition gate was set at 420–480 nm. Images were captured using the tile scan function in LAS X software to acquire a total of 9 tiles per tissue slice in a 4-slice Z-stack. The 3-dimensional volume of TUNEL positive cells was counted using LAS X software.
Alexafluor 546 goat anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AlexaFluor 546 goat anti-mouse secondary antibody is a fluorescently labeled antibody that binds to mouse primary antibodies. It is designed to be used in various immunodetection techniques, such as immunofluorescence and Western blotting, to visualize and detect target proteins.
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Lab products found in correlation
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (3 mentions)
C2 confocal microscope, by Nikon (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Lsm 710 meta, by Zeiss (2 mentions)
Mouse anti c myc antibody, by Merck Group (2 mentions)
Gene pulser 2, by Bio-Rad (2 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Primary mouse anti vinculin antibody, by Merck Group (2 mentions)
Alexafluor 647 chicken anti rat secondary antibody, by Thermo Fisher Scientific (2 mentions)
Click it plus tunel, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Ab90395, by Abcam (1 mentions)
Ab18147, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
A21052, by Thermo Fisher Scientific (1 mentions)
A 11030, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 633 goat antimouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
A12379, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
12 protocols using alexafluor 546 goat anti mouse secondary antibody
1
Quantification of Apoptosis in MHV-1 Pneumonia
2
Comprehensive Cell Cytoskeleton Staining
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After the end of the experiment, the scaffolds were fixed in 3% paraformaldehyde (Thermo Fisher Scientific) for 15 min and permeabilized in 1% Triton X-100 (Sigma-Aldrich) for 20 min. The actin cytoskeleton and cell nuclei were stained with Alexa Fluor 488 phalloidin (1:1000, A12379, Thermo Fisher Scientific) and DAPI (2 μg/ml, D1306, Invitrogen), respectively. Col-I primary antibody (ab90395, Abcam plc) was added to the medium at a dilution of 1:200 30 min before fixation to facilitate ECM staining, as described previously (44 (link)), and counterstained with AAlexa Fluor 633 goat anti-mouse secondary antibody (A-21052, Thermo Fisher Scientific) at 1:50. YAP was stained with YAP (63.7) primary mouse monoclonal antibody (sc-101199, Santa Cruz Biotechnology) at 1:200 and counterstained with Alexa Fluor 633 goat anti-mouse secondary antibody (A-21052, Thermo Fisher Scientific) at 1:50. α-SMA was stained with primary mouse anti–α-SMA antibody [0.N.5] (ab18147, Abcam plc) at 1:100 and counterstained with Alexa Fluor 546 goat anti-mouse secondary antibody (A-11030, Thermo Fisher Scientific) at 1:100. After washing with phosphate-buffered saline (PBS), primary and secondary antibodies were applied for 30 min each except for Col-I primary antibody, as mentioned above.
3
Quantifying Neuronal mRNA and Protein Levels
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mRNA measurements were performed with RNA isolated from cultured neurons at 14–15 days in vitro (DIV14-15). RT-PCR reactions were performed in triplicate with GAPDH as an internal control.
Mouse cortical neurons were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100, stained with anti-GAD65 (polyclonal; Sigma) and anti-MAP2 (monoclonal; Sigma) primary antibodies in PBS with 5% BSA, and visualized using Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 546 goat anti-mouse secondary antibodies (Molecular Probes). Images were acquired using a Nikon C2 confocal microscope equipped with a 60× oil-immersion objective. Identical settings were used for all samples in each experiment. We measured the average pixel intensities via manually tracing each dendrite, with a >2-fold background signal.
Mouse cortical neurons were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100, stained with anti-GAD65 (polyclonal; Sigma) and anti-MAP2 (monoclonal; Sigma) primary antibodies in PBS with 5% BSA, and visualized using Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 546 goat anti-mouse secondary antibodies (Molecular Probes). Images were acquired using a Nikon C2 confocal microscope equipped with a 60× oil-immersion objective. Identical settings were used for all samples in each experiment. We measured the average pixel intensities via manually tracing each dendrite, with a >2-fold background signal.
4
Quantifying Neuronal Protein Localization
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Various lentiviruses infected mouse cortical neurons were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100, incubated with anti-complexin (polyclonal; L669) and anti-vGlut1 (monoclonal; N28/9 (Neuromab)) primary antibodies in PBS with 5% BSA, washed, and stained with polyclonal anti-complexin and monocolonal anti-vGlut1 and visualized using Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 546 goat anti-mouse secondary antibodies (Molecular Probes). Images were acquired by using a Nikon C2 confocal microscope equipped with a 60× oil-immersion objective. We measured the average pixel intensities by manually tracing each dendrite, with a >2-fold background signal. Identical settings were applied to all samples in each experiment.
5
Immunolabeling of Neuronal Synapses
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Neurons were fixed in 4% paraformaldehyde at DIV13 and permeabilized with 0.2% Triton X-100, incubated with anti-synapsin1 (rabbit polyclonal antibody), and anti-MAP2 (Microtubule-associated protein 2, mouse monoclonal, Sigma M4403) primary antibodies in PBS with 5% BSA, washed, and visualized using Alexa Fluor-488 goat anti-rabbit and Alexa Fluor-546 goat anti-mouse secondary antibodies (Molecular Probes). Primary antibodies anti-synapsin1 were used to mark synapse of the cultured neuron. The antibodies anti-MAP2 were used to mark the dendrites of cultured neurons. Primary antibodies anti-vGult-1 were used to mark excitatory synapse and anti-GAD65 was used to mark inhibitory synapse. The antibodies anti-tubulin was used to mark the cultured neurons. Images were captured by a Nikon C2 confocal microscope equipped with a 60× oil-immersion objective. Synapse size and number were statistical by ImageJ (NIH).
6
Imaging Collybistin Localization in HEK293 Cells
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These were performed essentially as previously described (Long et al., 2016 (link)). HEK293 cells were co-transfected with the pRK5myc-hCB3SH3-R356Q construct at a 1:1 ratio with pEGFP-gephyrin using electroporation (Gene Pulser II, Bio-Rad). Cells were fixed after 24 h for 2 min in 4% (w/v) PFA in PBS. Immunostaining to detect collybistin was performed using a mouse anti-c-myc antibody (1:200, Sigma) and detected using an AlexaFluor 546 goat anti-mouse secondary antibody (1:600; Invitrogen). Counterstaining for cell nuclei was performed with DAPI (1:500; Life Technologies). Confocal microscopy was performed using a Zeiss LSM 710 META. All images were taken with a ×63 objective.
7
Immunofluorescent Profiling of Epithelial Markers
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Standard techniques for immunofluorescence were used to stain for pancytokeritin (mouse monoclonal anti-pancytokeratin FITC–conjugated antibody, Upstate Millipore Corp., USA), EpCAM (mouse monoclonal anti-CD326 Alexafluor® 488–conjugated antibody, Biolegend, USA), CA125 (mouse monoclonal anti-CA125 antibody, Abcam, USA, and Alexafluor® 546 goat anti-mouse secondary antibody, Invitrogen, USA), MOC-31 (mouse monoclonal anti-MOC-31 antibody, Dako, Germany, and Alexaflour® 596 goat anti-mouse secondary antibody, Invitrogen, USA), D2-40 (mouse monoclonal anti-D2-40 antibody, Dako, Germany, and goat anti-mouse Alexafluor® 596 secondary antibody, Invitrogen, USA) and vimentin (rabbit monoclonal anti-vimentin antibody, clone EPR3776, Abcam, USA, and Alexafluor® 488 goat anti-rabbit secondary antibody, Invitrogen, USA).
8
Whole-Mount Immunohistochemistry of Embryonic Nerves
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Whole-mount immunohistochemistry was performed as previously described [24 (link)]. Embryonic tissues were incubated with rabbit anti-GFP primary antibody (1:500; Life Technologies, Carlsbad, CA, USA) to detect the motor nerves. For detection of cranial nerves, embryonic heads were incubated with mouse monoclonal antibody 2H3 (1:500; Developmental Studies Hybridoma Bank, University of Iowa, IA, USA). Subsequently, tissues samples were incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (1:500; Invitrogen) and Alexa Fluor 546 goat anti-mouse secondary antibody (1:500; Invitrogen). After PBS washes, tissue samples were dehydrated through a methanol series (30%, 50%, 80%, and 100% for 30 min each), cleared with benzyl alcohol-benzyl benzoate (BABB), and imaged using confocal microscopy FV1200 (Olympus).
9
Immunostaining of Transfected HEK293 Cells
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These were performed essentially as previously described (Harvey et al., 2004 (link)). HEK293 cells were co-transfected with pRK5myc-hCB3SH3− wild-type, pRK5myc-hCB3SH3+ wild-type, pRK5myc-hCB3SH3− R338W, pRK5myc-hCB3SH3− R290H or pRK5myc-hCB3SH3− R356N/R357N constructs at a 1:1 ratio with pEGFP-gephyrin using electroporation (Gene Pulser II, Bio-Rad). Cells were fixed after 24 h for 2 min in 4% (w/v) PFA in PBS. Immunostaining to detect CB was performed using a mouse anti-c-myc antibody (1:200, Sigma) and detected using an AlexaFluor 546 goat anti-mouse secondary antibody (1:600; Invitrogen). Counterstaining for cell nuclei was performed with DAPI (1:500; Life Technologies). Confocal microscopy was performed using a Zeiss LSM 710 META. All images were taken with a × 63 objective.
10
Multimodal Imaging of Cell Adhesion
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