HEK-293 cultures expressing each chain individually were grown to confluency in Dulbecco’s modified Eagles medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO) in 4-well chamber slides. Cells were incubated overnight in media supplemented with 0.25 mM sodium ascorbate to facilitate collagen biosynthesis. Cells were fixed in 2% paraformaldehyde, permeabilized with 0.1% v/v Triton-X100 (Sigma), and blocked with 10% normal goat serum (Abcam, ab156046) in 1× PBS. Primary and secondary antibodies were added at the same concentration and incubation times as for the immunohistochemical studies (see Table 1 ).
Ab156046
Manufactured by Abcam
Sourced in United States
Ab156046 is a primary antibody. It is produced in rabbit and recognizes a specific target antigen. The antibody is purified using affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
D523 10, by Dojindo Laboratories (4 mentions)
Uplsapo30xs, by Olympus (4 mentions)
Cubic r, by Tokyo Chemical Industry (4 mentions)
Tcs sp8, by Leica (4 mentions)
Hc pl apo cs2, by Leica (4 mentions)
Fluoview fv1000, by Olympus (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (2 mentions)
Hyaluronidase, by Nacalai Tesque (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Tritc conjugated phalloidin, by Merck Group (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg antibody, by Jackson ImmunoResearch (1 mentions)
Ab97051, by Abcam (1 mentions)
Ab182422, by Abcam (1 mentions)
Mca1957, by Bio-Rad (1 mentions)
Ab142567, by Abcam (1 mentions)
10 protocols using ab156046
1
Immunostaining of HEK-293 Cells
2
Whole-Tissue Fluorescent Staining of Cochleae
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For the whole-tissue staining, we first removed cartilaginous capsule from the dissected cochleae and performed the fluorescence staining according to the previous study [51 (link)]. Briefly, the samples were fixed with 4% PFA in PBS overnight at 4°C and then blocked by incubation in 10% normal goat serum (Abcam, #ab156046) diluted in 0.1% Triton X-100/PBS (PBT) for 3 h at 37°C. The samples were treated with primary antibodies overnight at 4°C, washed in 0.1% PBT and subsequently treated with secondary antibodies conjugated to either Alexa Fluor 546 or Alexa Fluor 647 overnight at 4°C. DAPI (Dojindo Molecular Technologies, #D523-10, 1 : 200 dilution) was used for counterstaining of nucleus.
3
Immunohistochemistry of Epididymis
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Dissected epididymides were fixed with 4% PFA in PBS for 2 h at 4°C or for 20 min at 37°C, or with 2% TCA in Ca2+- and Mg2+-free PBS for 20 min at 4°C, depending on target epitopes. The samples were then blocked by incubation in 10% normal goat serum (NGS) (Abcam, ab156046) or 10% normal donkey serum (Abcam, ab166643) diluted in 0.1% Triton X-100/PBS, depending on the secondary antibody species, for 3 h at 37°C. The samples were treated with primary antibodies overnight at 4°C, and subsequently incubated with secondary antibodies conjugated to either Alexa Fluor 546 or Alexa Fluor 647 (1:1000, Thermo Fisher Scientific) overnight at 4°C. We used TRITC-conjugated phalloidin (0.3 μg/ml, Merck Millipore, FAK100) and Hoechst 33342 (5 μg/ml, Thermo Fisher Scientific, H3570) for the visualization of F-actin and DNA, respectively.
4
c-Fos Immunohistochemistry in Tissue Sections
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For Experiment C, sections were removed from TSB, washed in PBS, and submerged in 20% goat serum (ab156046, Abcam) for 2 h at room temperature. The sections were then washed three times with PBS-0.1% Triton X-100 Detergent (PBS-T). Then, they were incubated with a primary antibody against c-Fos (rabbit, #2250, 1:400; Cell Signaling Technology) at 4°C overnight. Subsequently, the sections were washed three times using PBS-T and then incubated in a secondary anti-rabbit IgG antibody conjugated with AlexaFluor 488 (Goat, #111-545-003, 1:400; Jackson ImmunoResearch) for 2 h at room temperature. The sections were washed three times with PBS, and were mounted on glass slides and sealed with coverslips and ProLong Diamond antifade mounting medium containing DAPI, as in Experiment A.
5
Tracheae Staining and Optical Clearing
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Staining and optical clearing of dissected tracheae were performed as described in a previous study (Hirashima and Adachi, 2015 (link)). Briefly, the samples were fixed with 4% PFA in PBS overnight at 4°C. For anti-SOX9 staining, the samples were incubated in 25 mg/mL hyaluronidase (Nacalai Tesque, #18240-36) for 1 h at 37°C, to digest hyaluronic acid. The samples were then blocked in 10% normal goat serum (Abcam, #ab156046) diluted in 0.1% Triton X-100/PBS (PBT) for 3 h at 37°C. The samples were treated with primary antibodies overnight at 4°C, washed in 0.1% PBT, and subsequently incubated in secondary antibodies conjugated to either Alexa Fluor 546 or Alexa Fluor 647 overnight at 4°C. DAPI was used for nuclear counterstaining (Dojindo Molecular Technologies, #D523-10, 1:200 dilution). The samples were mounted with 10 μL of 1% agarose gel onto a glass dish (Greiner Bio-One, #627871) for stable imaging. Then, the samples were immersed in CUBIC-R+ (Tokyo Chemical Industry Co., # T3741) solution for optical clearing. Images were acquired using the confocal laser scanning platform Leica TCS SP8 equipped with the hybrid detector Leica HyD, using a ×40 objective lens (NA = 1.3, WD = 240 μm, HC PL APO CS2, Leica) and the Olympus FluoView FV1000 with a ×30 objective lens (NA = 1.05, WD = 0.8 mm, UPLSAPO30XS, Olympus).
6
Macrophage Infiltration Analysis in PEG Hydrogel Implants
7
BrdU Immunolocalisation in Carapace Epithelia
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BrdU immunolocalisation was assayed in cultured intact carapace-epithelia (n = 5) following BrdU incorporation in vitro over 48 h in a 10 μM BrdU (ab142567; Abcam) labelling solution. The samples were prepared, fixed and permeabilised as described above for EdU incorporation, then incubated in 1 M HCl for 30 min at room temperature. The samples were then blocked in 5% normal goat serum (ab156046; Abcam) in PBS for 1 h, incubated in rabbit polyclonal anti-BrdU (1/100 dilution, Abcam, ab152095) overnight at 4°C and then rinsed three times in 1× PBS. After an incubation for 2 h in the dark at room temperature in goat anti-rabbit IgG H&L conjugated to Alexa Fluor 555 (1/1000 dilution, ab150086, Abcam), the samples were mounted in ProLong Gold Anti-fade mountant with DAPI (Thermo Fisher Scientific) and visualised using fluorescent microscopy. Percentage cell proliferation was quantified.
8
Cochlear Immunostaining and Optical Clearing
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The cochleae were gently freed from the capsule, and the staining and clearing were performed according to an earlier study (Hirashima and Adachi, 2015 (link)). Briefly, the samples were fixed with 4% PFA in PBS overnight at 4°C and then blocked by incubation in 10% normal goat serum (Abcam, #ab156046) diluted in 0.1% Triton X-100/PBS (PBT) for 3 hr at 37°C. The samples were treated with primary antibodies overnight at 4°C, washed in 0.1% PBT, and subsequently treated with secondary antibodies conjugated to either Alexa Fluor 546 or Alexa Fluor 647 overnight at 4°C. For counter staining of nucleus, we used DAPI (Dojindo Molecular Technologies, #D523-10, 1:200 dilution). The samples were mounted with 10 µL of 1% agarose gel onto a glass-based dish (Greiner Bio-One, #627871) for stable imaging. Then, the samples were immersed with the CUBIC-R+ (Tokyo Chemical Industry Co., #T3741) solution for optical clearing. Images were acquired using the confocal laser scanning platform Leica TCS SP8 equipped with the hybrid detector Leica HyD with the ×40 objective lens (NA = 1.3, WD = 240 μm, HC PL APO CS2, Leica) and the ×20 objective lens (NA = 0.75, WD = 680 µm, HC PL APO CS2, Leica) and the Olympus FluoView FV1000 with the ×30 objective lens (NA = 1.05, WD = 0.8 mm, UPLSAPO30XS, Olympus).
9
Cochlear Tissue Clearing and Imaging
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The cochleae were gently freed from the capsule and the staining and clearing were performed according to an earlier study (Hirashima and Adachi, 2015) (link). Briefly, the samples were fixed with 4% PFA in PBS overnight at 4°C and then blocked by incubation in 10% normal goat serum (Abcam, #ab156046) diluted in 0.1% Triton X-100/PBS (PBT) for 3 h at 37°C. The samples were treated with primary antibodies overnight at 4°C, washed in 0.1% PBT, and subsequently treated with secondary antibodies conjugated to either Alexa Fluor 546 or Alexa Fluor 647 overnight at 4°C. For counter staining of nucleus, we mixed Hoechst 33342 (5 μg/ml, Dojindo Molecular Technologies, #H342-10) or DAPI (Dojindo Molecular Technologies, #D523-10, 1:200 dilution) . The samples were mounted with 10 µL of 1% agarose gel onto a glass-based dish (Greiner Bio-One, #627871) for stable imaging. Then, the samples were immersed with the BABB (benzyl-alcohol and benzyl-benzoate, 1:2, #04520-96 and # 04601-65, Nacalai Tesque) solution or CUBIC-R+ (Tokyo Chemical Industry Co., # T3741) solution for optical clearing. Images were acquired using the confocal laser scanning platform Leica TCS SP8 equipped with the hybrid detector Leica HyD with the ×40 objective lens (NA = 1.3, WD = 240 μm, HC PL APO CS2, Leica) and the Olympus FluoView FV1000 with the ×30 objective lens (NA = 1.05, WD = 0.8 mm, UPLSAPO30XS, Olympus).
10
Staining and Optical Clearing of Tracheae
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Staining and optical clearing of dissected tracheae were performed as described in a previous study (Hirashima and Adachi, 2015) (link). Briefly, the samples were fixed with 4% PFA in PBS overnight at 4°C. For anti-SOX9 staining, the samples were incubated in 25 mg/mL hyaluronidase (Nacalai Tesque, #18240-36) for 1 h at 37°C, to digest hyaluronic acid. The samples were then blocked in 10% normal goat serum (Abcam, #ab156046) diluted in 0.1% Triton X-100/PBS (PBT) for 3 h at 37°C. The samples were treated with primary antibodies overnight at 4°C, washed in 0.1% PBT, and subsequently incubated in secondary antibodies conjugated to either Alexa Fluor 546 or Alexa Fluor 647 overnight at 4°C. DAPI was used for nuclear counterstaining (Dojindo Molecular Technologies, #D523-10, 1:200 dilution). The samples were mounted with 10 µL of 1% agarose gel onto a glass dish (Greiner Bio-One, #627871) for stable imaging. Then, the samples were immersed in CUBIC-R+ (Tokyo Chemical Industry Co., # T3741) solution for optical clearing. Images were acquired using the confocal laser scanning platform Leica TCS SP8 equipped with the hybrid detector Leica HyD, using a ×40 objective lens (NA = 1.3, WD = 240 μm, HC PL APO CS2, Leica) and the Olympus FluoView FV1000 with a ×30 objective lens (NA = 1.05, WD = 0.8 mm, UPLSAPO30XS, Olympus).
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