We used an antibody for RNA polymerase II as a previous report (Shirae-Kurabayashi et al, 2011 (link)); CTD-pSer2 (Abcam, ab5095, 1:500 dilution). We followed the previously described protocol (Ohta and Satou, 2013 (link)) with slight modification. A rabbit anti-human phospho-JAK2 (Y931) antibody (Thermo Fisher Scientific, PA5-104704) was used as a primary antibody, 1/500 in Can Get Signal Immunostain Solution A (TOYOBO). The antibody was detected by an anti-rabbit-HRP goat antibody 1/500 in Can Get Signal Immunostain Solution A (TOYOBO), and by Tyramide Signal Amplification (Perkin Elmer) as previously described (Ohta and Satou, 2013 (link)).
Can get signal immunostain solution a
Manufactured by Toyobo
Sourced in Japan, United States
Can Get Signal Immunostain Solution A is a laboratory reagent used for immunostaining. It is designed to facilitate the detection and visualization of target proteins or antigens in biological samples during immunohistochemical or immunofluorescence analysis.
Automatically generated - may contain errors
Lab products found in correlation
Image it fx signal enhancer, by Thermo Fisher Scientific (2 mentions)
Bz 9000, by Keyence (2 mentions)
Hybrid cell count software bz h3c, by Keyence (2 mentions)
Blocking one, by Nacalai Tesque (2 mentions)
Tyramide signal amplification, by PerkinElmer (2 mentions)
Bz 8000 confocal microscope, by Keyence (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Epr2417y, by Abcam (1 mentions)
Lsm image browser software, by Zeiss (1 mentions)
Doublecortin, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Active caspase 3, by Abcam (1 mentions)
Bz x700 microscope, by Keyence (1 mentions)
Histofine simple stain max po, by Nichirei Biosciences (1 mentions)
Bz x700, by Keyence (1 mentions)
Deltavision microscope system, by GE Healthcare (1 mentions)
27 protocols using can get signal immunostain solution a
1
Phospho-JAK2 (Y931) Immunodetection
2
Immunocytochemical Staining of Neuronal Cells
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The cells were plated on Matrigel (CORNING, Corning, NY, USA) coated NUNC Thermanox plastic coverslip (Thermo Fisher Scientific) in a 24-well plate. The cells were fixed with 4% PFA (Nacalai Tesque) for 15 min. at room temperature (R.T.). After washing thrice with PBS, the cells were incubated with 0.1% Triton-X100/PBS for 15 min. at R.T., rinsed with PBS thrice again, and treated with Blocking One (Nacalai Tesque) for 1 hr. at R.T. The blocking buffer was replaced with primary antibody solution (mouse anti-beta III tubulin antibody: T8660 (Sigma Chemical) 1/500 diluted with Can Get Signal immunostain Solution A (TOYOBO, Osaksa, Japan)), and the specimens were incubated for O/N (overnight) at 4 deg C. After washing thrice with PBS, secondary antibody solution (Goat anti-Rabbit igG (H + L) Cross-Absorbed Secondary Antibody, Alexa Fluor 568: A-11031 (Thermo Fisher Scientific) 1/500 diluted with Can Get Signal immunostain Solution A (TOYOBO)) was applied for 1 hr. at R.T. in the dark. The cells were washed with PBS thrice, and incubated with 1/10000 DAPI solution/PBS (DOJINDO, Kumamoto, Japan) for 10 min. at R.T. in the dark. After washing thrice with PBS, the coverslip was mounted onto a slide glass with antifade mounting reagents (Thermo Fisher Scientific).
3
Immunofluorescence Staining of ATF3 in Colon Tissue
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Colon sections (5 μm) were prepared from the paraffin blocks of two CD and two UC patients. After deparaffinization, antigen retrieval was performed in Tris-EDTA buffer (10mM Tris, 1mM EDTA, 0.05% Tween 20, pH9.0) at 95°C for 20 min, followed by blocking with Image-iT FX Signal Enhancer (Life technologies) at room temperature for 20 min. Colon sections were incubated with anti-ATF3 antibodies (clone 44C3a, Abcam) (1/100), which had been diluted with Can Get Signal immunostain solution A (Toyobo), for 1 h at 37°C and then washed three times with phosphatebuffered saline (PBS). Alexa Fluor 488 goat antimouse IgG (Molecular Probes) were used as secondary antibodies (1/200), which had been diluted with Can Get Signal immunostain solution A (Toyobo), for 30 min at 37°C. Slides were then washed three times with PBS, fixed with ProLong Diamond Antifade Mountant, stained with 4',6-diamidino-2-phenylindole (Molecular Probes), and visualized using an Olympus BX51 microscope and CCD camera (DP73; Olympus).
4
Antibody-based detection of phosphorylated proteins
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We used an antibody for RNA polymerase II as per a previous report 17 ; CTD-pSer2 (Abcam, ab5095, 1:500 dilution). We followed the previously described protocol 35 with slight modification. A rabbit anti human phospho-JAK2 (Y931) antibody (Thermo Fisher Scientific, PA5-104704) was used as a primary antibody, 1/500 in Can Get Signal Immunostain Solution A (TOYOBO). The antibody was detected by an anti-rabbit-HRP goat antibody 1/500 in Can Get Signal Immunostain Solution A (TOYOBO), and by Tyramide Signal Amplification (Perkin Elmer) as previously described 35 .
In situ hybridization DNA fragments were amplified by PCR with exTaq-HS (Takara Bio) and Phusion HF (New England Biolabs) DNA polymerases from Ciona genomic DNA or cDNA. The primers that we used were summarized in Supplemental table S1. The amplicons were subcloned into TOPO vectors (life technologies). DIG or fluorescein labeled RNA probes were synthesised by T7 and sp6 RNA polymerases (Roche) from template DNA plasmid digested by NotI or SpeI (New England Biolabs), and were cleaned by RNeasy mini kit (QIAGEN). We followed the protocol for in situ hybridization described before 35, 41 . We detected fluorescein and DIG probes using TSA plus (Perkin Elmer) green (FP1168) and red (FP1170), respectively. Primer sequences are provided in Supplemental Table S1.
In situ hybridization DNA fragments were amplified by PCR with exTaq-HS (Takara Bio) and Phusion HF (New England Biolabs) DNA polymerases from Ciona genomic DNA or cDNA. The primers that we used were summarized in Supplemental table S1. The amplicons were subcloned into TOPO vectors (life technologies). DIG or fluorescein labeled RNA probes were synthesised by T7 and sp6 RNA polymerases (Roche) from template DNA plasmid digested by NotI or SpeI (New England Biolabs), and were cleaned by RNeasy mini kit (QIAGEN). We followed the protocol for in situ hybridization described before 35, 41 . We detected fluorescein and DIG probes using TSA plus (Perkin Elmer) green (FP1168) and red (FP1170), respectively. Primer sequences are provided in Supplemental Table S1.
5
Histopathological and Immunostaining Analysis of Tissue Samples
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For histopathological examination, the tissue were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin (JUNSEI, Cat. #58290-1501) using standard procedures. Sections (3 μm) were stained with Hematoxylin & Eosin.
For ANXA10 immunostaining, cattle abomasum samples were collected into cryotube and snap frozen with liquid nitrogen. Tissues were trimmed using cryostat blade (Leica, Cat. #14035838926) and then embedded in Tissue-Tek O.C.T. compound (Sakura, Cat. #4583) and frozen. Section (15 μm) was fixed in 4% PFA at 4 °C for 15 min. Mice stomach tissue were fixed with 4% PFA, and the tissues were rinsed with PBS, 30% sucrose/PBS and embedded in O.C.T compound. Sections (20 μm) were washed with 0.05% Triton x100 (Wako, Cat. #168-11805) in PBS. For ANXA10 immunostaining, the sections were immunostained with anti-Annxin A10 antibody (R&D, Cat. #AF3544, 5 μg/ml) in Canget signal immunostain solution A (TOYOBO, Cat. #NKB-401) for 1 h. For platelet thrombi, the sections were immunostained with anti-integrin beta 3 [EPR2417Y] (abcam, Cat. #ab75872, 1:250) [40 (link)].
For ANXA10 immunostaining, cattle abomasum samples were collected into cryotube and snap frozen with liquid nitrogen. Tissues were trimmed using cryostat blade (Leica, Cat. #14035838926) and then embedded in Tissue-Tek O.C.T. compound (Sakura, Cat. #4583) and frozen. Section (15 μm) was fixed in 4% PFA at 4 °C for 15 min. Mice stomach tissue were fixed with 4% PFA, and the tissues were rinsed with PBS, 30% sucrose/PBS and embedded in O.C.T compound. Sections (20 μm) were washed with 0.05% Triton x100 (Wako, Cat. #168-11805) in PBS. For ANXA10 immunostaining, the sections were immunostained with anti-Annxin A10 antibody (R&D, Cat. #AF3544, 5 μg/ml) in Canget signal immunostain solution A (TOYOBO, Cat. #NKB-401) for 1 h. For platelet thrombi, the sections were immunostained with anti-integrin beta 3 [EPR2417Y] (abcam, Cat. #ab75872, 1:250) [40 (link)].
6
Immunofluorescence Microscopy of Primary Cilia
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Immunofluoresence microscopy was performed using confocal microscopy (LSM510 META; Carl Zeiss) equipped with a microscope (Axiovert 200 M; Carl Zeiss), a plan Apochromat × 100/1.4 NA oil immersion lens and LSM image Browser software (Carl Zeiss) as described29 (link) with slight modifications. The list of antibodies with source and conditions of indirect immunofluorescence is shown in Supplementary Table 2 . In some immunofluorescence experiments, we used Can Get Signal immunostain Solution A (TOYOBO). For induction of primary cilia, RPE1 and IMR-90 cells were seeded on sterile coverslips, grown for 1 day and then subjected to serum starvation. For detection of primary cilia, cells were placed on ice for 20–30 min before fixation with cold methanol, and stained with anti-acetylated tubulin antibody29 (link). BrdU incorporation was evaluated using DNA Replication Assay Kit (Millipore). Quantification of fluorescence intensity was performed using ImageJ 1.45r software.
7
Immunofluorescence Staining of Formalin-Fixed Tissues
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For the staining of tissues, mice were sacrificed and transcardially perfused with saline, followed by 10% neutral buffered formalin (pH 7.4). Formalin-fixed tissues were then embedded in paraffin and sectioned. For immunofluorescence staining, paraffin-embedded sections were deparaffinized, and blocking was performed in Image-iT FX Signal Enhancer (Life Technologies) for 30 min. Primary antibodies were obtained from Abcam (active caspase-3, doublecortin and Choline Acetyltransferase). Secondary antibodies were obtained from Thermo (Alexa Fluor 488 Goat Anti-rabbit IgG (H + L)). The antibodies were diluted with Can Get Signal Immunostain Solution A (TOYOBO). After the samples had been mounted on a slide with a cover glass, they were observed under a Keyence BZ-9000 fluorescence microscope.
8
Immunofluorescent Laminin and Dystrophin Detection
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De-paraffinized sections were digested with proteinase (Dako Retrieval Solution Ready-to-Use; Dako, Glostrup, Denmark) for 20 min, and the sections were treated overnight at 4°C with the antibodies in Can Get Signal immunostain Solution A (Toyobo, Osaka, Japan); goat anti-mouse Laminin α-1 polyclonal antibody (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, California, USA) and rabbit anti-mouse Dystrophin polyclonal antibody (1:100 dilution; Santa Cruz Biotechnology). The sections were then incubated with the secondary antibodies; chicken anti-goat immunoglobulin Alexa Fluor 488 (1:100 dilution; Life Technologies, Carlsbad, California, USA), and chicken anti-rabbit immunoglobulin Alexa Fluor 594 (1:100 dilution; Life Technologies) for 60 min at room temperature. The nucleus was stained with DAPI, and images were obtained using a BZ-X700 microscope (Keyence, Osaka, Japan).
9
Immunohistochemical Analysis of Myogenic Markers
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To investigate the expression of myogenic markers, we performed an immunohistochemical assessment of MyoD and myogenin. Formalin‐fixed and paraffin‐embedded muscle tissue were pretreated with proteinase K, quenched with 0.05% H₂O₂, and incubated overnight at 4°C in Can Get Signal Immunostain Solution A (Toyobo, Osaka, Japan) with the following primary antibodies: mouse anti‐MyoD antibody (Santa Cruz Biotechnology, sc‐32 758, 1:100) and mouse anti‐myogenin antibody (Santa Cruz Biotechnology, sc‐52 903, 1:100). Following washes twice with Phosphate Buffered Saline, the sections were incubated with peroxidase‐conjugated anti‐mouse antibody (Histofine Simplestain MAX‐PO; Nichirei, Tokyo, Japan; code 424131) for 30 minutes. Following adequate diaminobenzidine staining and counterstaining with haematoxylin, tissue images were acquired using a fluorescence microscope BZ‐X700 (Keyence) (×400), and the positive area was automatically quantified using the Hybrid Cell Count BZ‐H3C software (Keyence).
10
Immunostaining of p62 in Murine Fibroblasts
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Murine embryonic fibroblast cells grown in a glass‐bottom dish were fixed with 4% PBS for 15 min at room temperature. After washing with 0.1% Tween 20 in PBS (PBST), cells were permeabilized with 0.5% Triton X‐100 in PBS for 5 min at room temperature and washed with PBST three times. The cells were then blocked with Blocking One solution (03953‐95; Nacalai Tesque) for 30 min at 4 °C, washed once with PBST, and incubated with a rabbit polyclonal anti‐p62 antibody (P0067; Sigma‐Aldrich) in Can Get Signal Immunostain Solution A (NKB‐501; Toyobo, Osaka, Japan) for 2 h, followed by extensive washes and incubation with Cy3‐conjugated donkey anti‐rabbit IgG antibody (AP182C; Life Technologies, Carlsbad, CA, USA) for 1 h. After washing with PBST three times, cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and subjected to FM. For observation, an oil‐immersion objective lens (PLAPON60xOSC/NA1.40; Olympus) on the DeltaVision microscope system (GE Healthcare Life Sciences) was used as previously described 25 .
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