Deparaffinized sections were boiled for 20 min in 10 mM citrate buffer (pH 6) (Genemed Biotechnologies) for antigen retrieval, rinsed in distilled water, and then blocked with 3% normal goat serum (Vector Laboratories) in PBS for 1 h at room temperature. The sections were incubated with primary antibodies in PBS or Can Get Signal immunostain solution (Toyobo, Osaka, Japan) for 1 h at room temperature. The primary antibodies were as follows: rabbit anti-GFAP, rabbit anti-Iba1, rabbit anti-NeuN, and rabbit anti-olfactory marker protein (OMP) (#ab183947, 1:1,000; Abcam, Cambridge, UK). After washing in PBS, the sections were incubated with Alexa Fluor 488- or 594-conjugated goat anti-rabbit IgG antibodies (#A31627 and #A-11012, 1:1,000; Thermo Fisher Scientific, Waltham, MA, USA) in PBS or Can Get Signal immunostain solution for 1 h at room temperature, washed in PBS, and mounted using VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories). All images were captured and analyzed using the ECLIPSE Ti confocal microscope (Nikon Instruments) with NIS-Elements AR imaging software version 4.00.06 (Nikon Instruments). The number of OMP-positive cells was measured in all bilateral subareas, and their mean values were calculated for each animal.
Can get signal immunostain solution
Manufactured by Toyobo
Sourced in Japan
Can Get Signal immunostain solution is a laboratory reagent used in immunohistochemistry (IHC) applications. It is a ready-to-use solution designed to enhance the visualization and detection of target proteins or antigens in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Ab183947, by Abcam (1 mentions)
Nis elements ar imaging software, by Nikon (1 mentions)
Vectashield vibrance antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Eclipse ti confocal microscope, by Nikon (1 mentions)
Fitc conjugated anti mouse cd326 epcam mab, by BioLegend (1 mentions)
Bz x800 microscope, by Keyence (1 mentions)
Bz x800 analyzer software, by Keyence (1 mentions)
Lsm710 780, by Zeiss (1 mentions)
Plan apochromat 63 1.40 oil lens, by Zeiss (1 mentions)
Fluorosave, by Merck Group (1 mentions)
αplan fluar 100 1.45 oil lens, by Zeiss (1 mentions)
Lsm 880 airyscan, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Lsm710 confocal laser fluorescence microscope, by Zeiss (1 mentions)
Hoechst 33342, by Dojindo Laboratories (1 mentions)
Triton 100, by Merck Group (1 mentions)
Fitc anti mouse cd326 epcam, by BioLegend (1 mentions)
Alexa fluor 594 conjugated anti mouse igg h l, by Cell Signaling Technology (1 mentions)
7 protocols using can get signal immunostain solution
1
Immunostaining Protocol for Olfactory Markers
2
Multicolor Immunofluorescence Staining of Salivary Gland
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Frozen sections of salivary gland tissue were fixed with methanol/acetone (1:1), blocked using 5% normal goat serum (WAKO)/0.3% Triton X-100 (Sigma) in phosphate-buffered saline (PBS), and stained with FITC-conjugated anti-mouse CD326 (EpCAM) mAb (118207, BioLegend), Alexa Fluor 647-conjugated anti-mouse CD4 mAb (100426, BioLegend), anti-mouse CD153 mAb (14-1531-85, Thermo Fisher Scientific), and an anti-mouse CXCL13 polyclonal antibody (PA1-29046, Thermo Fisher Scientific). Alexa Fluor 555-conjugated anti-rat IgG (H+L) (4417) and Alexa Fluor 594-conjugated anti-rabbit IgG (H+L) (8889), which were obtained from Cell Signaling Technology, were used as the secondary antibody. These antibodies were diluted with the Can Get Signal immunostain solution (Toyobo, Osaka, Japan). After washing 3 times with PBS, nuclear DNA was stained with ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were acquired using the KEYENCE BZ-X800 microscope (Osaka, Japan), and processed with the full focus function of the BZ-X800 Analyzer software (Itasca, IL, USA)unless otherwise stated.
3
Immunostaining of Adherent and Spheroid Cells
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Cells grown on cover glasses were fixed with 1% (wt/vol) PFA in the medium at RT for 10 min. In the case of sphere cultures, 2% (wt/vol) PFA was used for fixation. Cells were made permeable with 0.5% Triton X-100 in TBS for 20 min. The samples were blocked with 3% (wt/vol) BSA and 10% (vol/vol) goat serum in TBS containing 0.1% Triton X-100 (TBS-T) and incubated with primary antibodies in the Can Get Signal immunostain solution (TOYOBO) at RT for 2 h. After washes, the cells were incubated with fluorescence-labeled secondary antibodies for 1 h. After further washes, cells were incubated with fluorescence-labeled phalloidin (Molecular Probes) for 30 min and subsequently mounted using FluoroSave (EMD Millipore). Images of cells were obtained using a laser-scanning confocal microscope LSM710/780 (ZEISS) equipped with an αPlan-FLUAR 100×/1.45 oil lens or Plan-Apochromat 63×/1.40 oil lens (ZEISS) at RT. Z-stack images were taken at every 0.3 µm. For observation of the lateral views with higher resolution, images were obtained by the laser-scanning confocal microscope LSM880-Airyscan (ZEISS) equipped with an αPlan-FLUAR 100×/1.45 oil lens at RT. Z-stack images were taken at every 0.17 µm and subjected to Airyscan super-resolution mode processing. Images were processed using ZEN software (ZEISS) and Photoshop CS5 (Adobe Systems).
4
Nrf2 Activation in Fibroblasts by Mild PAM
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Fibroblasts (6 × 104 cells) were seeded on collagen-coated coverslips (12 mm in diameter) in a 4-well culture plate (Nunc 176740) and cultured for 24 h in a CO2 incubator. After the treatment with mild PAM (500 μl) for 6 h in a CO2 incubator, cells were washed with PBS followed by fixation with 3% paraformaldehyde, permeabilization with 0.1% Triton X-100, and blocking with 3% BSA solution. Cells were then incubated for 1 h with an anti-Nrf2 antibody (1:50) diluted with Can Get Signal Immunostain solution (Toyobo), followed by an incubation for 1 h with Alexa Fluor 488 goat anti-rabbit IgG (1:400). After the labeling of cell nuclei with Hoechst 33342 (1:1,000, Dojindo, Kumamoto, Japan), cells were washed and visualized under the LSM710 confocal laser fluorescence microscope (Carl Zeiss, Gottingen, Germany).
5
Immunofluorescence Analysis of Lacrimal Gland
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Frozen sections of lacrimal gland tissue were fixed with methanol/acetone (1:1), blocked using 5% normal goat serum (WAKO)/0.3% Triton×-100 (Sigma) in phosphate-buffered saline (PBS), and stained with FITC anti-mouse CD326 (EpCAM) (118207, BioLegend), AdipoR2 (sc-514045), and PPARγ (2443) antibodies. Alexa Fluor594-conjugated anti-mouse IgG (H+L) (8890, Cell Signaling Technology) and Alexa Fluor 555-conjugated anti-Rabbit IgG (H+L) (A21428, Thermo Fisher Scientific) were used as secondary antibodies. These antibodies were diluted with Can Get Signal immunostain solution (Toyobo). After washing 3 times with PBS, nuclear DNA was stained with ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). Sections were observed using a fluorescence microscope (KEYENCE) at a magnification of 400× or 1000×.
6
Immunohistochemical Staining of Mouse Tissues
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The mice were anesthetized, perfused with phosphate buffered saline (PBS) and subsequently with 4% formaldehyde in PBS. Tissues were excised and immediately fixed with 4% formaldehyde in PBS overnight and embedded in paraffin. Sections were cut, deparaffinized, permeabilized with 0.1% Triton and heated in 10 mM citrate buffer (pH 6.0) using a microwave for antigen retrieval. Diaminobenzidine (DAB) staining was performed using a VECTASTAIN Elite ABC Kit (Vector Labs, Burlingame, CA, USA) and ImmPACT DAB (Vector Labs) according to the manufacturer’s instructions. For fluorescent immunostaining, sections were blocked with 3% goat serum in PBS for 1 h, and then incubated with primary antibodies diluted in Can Get Signal Immunostain Solution (Toyobo, Osaka, Japan) overnight at 4 °C. Sections were then incubated with secondary antibodies conjugated with fluorescent dye for 1 h, and then mounted using a Vector TrueVIEW Autofluorescence Quenching Kit (Vector). The primary antibodies used were anti-myosin (skeletal, slow) (1:400, M8421, SIGMA), anti-ChAT (1:5000 for DAB staining, provided by Dr. Hidemi Misawa22 (link)), anti-ChAT (1:1000 for fluorescent immunostaining, MA5-31383, Invitrogen), anti-TFG (1:100, ab156866, Abcam) and anti-OPN (1:100, sc-21742, Santa Cruz).
7
PARP-1 Activity Evaluation by Immunostaining
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PARP-1 activity was detected by the immunostaining of polyADPR, a product of this enzymatic reaction. A549 cells were seeded on collagen-coated coverslips (12 mm in diameter) placed in a 4-well culture plate (seeded at 1 × 105 cells/well), cultured for 24 h in a CO2 incubator, and then used in experiments. After the treatment of cells with PAM (400 μL) in a CO2 incubator for the indicated hours, the cells were washed with PBS followed by fixation with 3% paraformaldehyde, permeabilization with 0.1% Triton X-100, and then blocked with 3% BSA solution. Cells were incubated for 1 h with an anti-polyADPR (10H) mouse IgG monoclonal antibody (1:50; Immuno-Biological Laboratories, Fujioka, Japan) diluted with Can Get Signal Immunostain solution (Toyobo, Ootsu, Japan). After the cells had been washed with PBS, they were incubated for 1 h with Alexa Fluor 488 goat anti-mouse IgG antibody (1:400; Invitrogen) diluted with Can Get Signal Immunostain solution. After the labeling of cell nuclei with Hoechst 33342 (1:1000), cells were washed and visualized under the LSM710 confocal laser fluorescence microscope.
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