For immunofluorescence, deparaffinized sections were incubated in the “antigen unmasking solution” (10 mM tri-sodium citrate, 0.05% Tween-20) for 10 min at 75 °C, and treated with a blocking solution (3% BSA in PBS) for 30 min. Next, the primary antibody (anti-MHC-I, mouse monoclonal A4.951, Hybridoma Bank; anti-phospho αB-crystallin S59, rabbit polyclonal ab5577, Abcam; anti-β-actin, rabbit polyclonal ab8227, Abcam) diluted 1:50, was applied, and the sections were incubated in a humidified chamber overnight at 4 °C. Then, the sections were incubated for 1 h at room temperature with a conjugated secondary antibody (anti-rabbit IgG–FITC antibody produced in goat, F0382, Sigma-Aldrich; anti-mouse IgG-TRITC antibody produced in goat, T5393, Sigma-Aldrich). Nuclei were stained with Hoechst Stain Solution (1:1,000, Hoechst 33258, Sigma-Aldrich). The slides were treated with PermaFluor Mountant (Thermo Fisher Scientific) and cover slipped. The images were captured using a Leica Confocal Microscope TCS SP8 (Leica Microsystems). Staining intensity for phospho-αB-crystallin of different skeletal muscle fibers was expressed as the mean pixel intensity (PI) normalized to the CSA (cross-sectional area expressed in pixel) using the software Leica application suite advanced fluorescence software as previously described [35 (link)].
Ab5577
Manufactured by Abcam
Sourced in United States
Ab5577 is a laboratory equipment product. It is a primary antibody that can be used for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst stain solution, by Merck Group (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
F0382, by Merck Group (1 mentions)
T5393, by Merck Group (1 mentions)
Permafluor mountant, by Thermo Fisher Scientific (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab16284, by Abcam (1 mentions)
Ab421, by Abcam (1 mentions)
Ab222885, by Abcam (1 mentions)
3 3 diaminobenzidine (dab), by Thermo Fisher Scientific (1 mentions)
Ab34703, by Abcam (1 mentions)
Ab108400, by Abcam (1 mentions)
Ta 060 qhdx, by Thermo Fisher Scientific (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
A21207, by Thermo Fisher Scientific (1 mentions)
3 protocols using ab5577
1
Immunofluorescence analysis of muscle fibers
2
Immunohistochemical analysis of CRYAB phosphorylation
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Myocardial tissues were fixed in 4% paraformaldehyde for 72 h and then were embedded in paraffin and sectioned into 5-μm slices. The paraffin sections were subjected to haematoxylin-eosin (HE) staining and were analyzed under an optical microscope at 400 × magnification.
Paraffin sections were deparaffinized and immersed in distilled water. There after, the sections were blocked with 3% hydrogen peroxide at room temperature for endogenous peroxidase ablation for 15 min, followed by blockade in normal goat serum at room temperature for 20 min. After discarding the goat serum, the sections were incubated with rabbit anti-CRYAB phosphor-S59 (ab5577; Abcam, United States) at 4°C overnight, followed by incubation with goat anti-rabbit IgG (ab16284; Abcam, United States) for 1 h at room temperature. After colouration with 3, 3-diaminobenzidin (DAB) and staining with haematoxylin, the sections underwent dehydration, clearing and mounting with neutral gums. Images were visualized and collected under an optical microscope at 400 × magnification.
Paraffin sections were deparaffinized and immersed in distilled water. There after, the sections were blocked with 3% hydrogen peroxide at room temperature for endogenous peroxidase ablation for 15 min, followed by blockade in normal goat serum at room temperature for 20 min. After discarding the goat serum, the sections were incubated with rabbit anti-CRYAB phosphor-S59 (ab5577; Abcam, United States) at 4°C overnight, followed by incubation with goat anti-rabbit IgG (ab16284; Abcam, United States) for 1 h at room temperature. After colouration with 3, 3-diaminobenzidin (DAB) and staining with haematoxylin, the sections underwent dehydration, clearing and mounting with neutral gums. Images were visualized and collected under an optical microscope at 400 × magnification.
3
Immunohistochemical Analysis of Lens Proteins
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After deparaffinization and rehydration, the sections were boiled for 10 min in diluted with ddH 2 O from ×100 to ×1 antigen repair solution (MXB Biotechnologies, China) and blocked with 5% BSA for 1 h. Anti-Capn1 monoclonal
antibody (ab108400, ABCAM, USA, 1:100), Anti-Tgm2 polyclonal antibody (ab421, ABCAM, USA, 1:500), Anti-Camk2b polyclonal antibody (ab34703, ABCAM, USA, 1:200), Anti-Gja8 polyclonal antibody (ab222885, ABCAM, USA, 1:100); Anti-Cryab polyclonal antibody (ab5577, ABCAM, USA, 1:200), anti-Cryba1 polyclonal antibody (PA5-71690, ThermoFisher Scientific, USA, 1:500), anti-Crybb1 polyclonal antibody (bs-12582R, Bioss Antibodies, China, 1:500), and anti-Crybb3 polyclonal antibody (21009-1-AP, Proteintech Systems, USA, 1:100) were used as primary antibody. Anti-Rabbit antibody488 (A-21206, Invitrogen, USA), and anti-Rabbit antibody594 (A-21207, Invitrogen, USA) as secondary antibodies were incubated at room temperature for 2 h. Cell nuclei were counterstained with DAPI. DAB (TA-060-QHDX Ther-moFisher Scientific, USA) was used for color development followed by hematoxylin counterstaining in immunohistochemistry assay. Three mice/six lenses from each group were evaluated to compare these proteins expression of these two groups and repeated 3 times.
antibody (ab108400, ABCAM, USA, 1:100), Anti-Tgm2 polyclonal antibody (ab421, ABCAM, USA, 1:500), Anti-Camk2b polyclonal antibody (ab34703, ABCAM, USA, 1:200), Anti-Gja8 polyclonal antibody (ab222885, ABCAM, USA, 1:100); Anti-Cryab polyclonal antibody (ab5577, ABCAM, USA, 1:200), anti-Cryba1 polyclonal antibody (PA5-71690, ThermoFisher Scientific, USA, 1:500), anti-Crybb1 polyclonal antibody (bs-12582R, Bioss Antibodies, China, 1:500), and anti-Crybb3 polyclonal antibody (21009-1-AP, Proteintech Systems, USA, 1:100) were used as primary antibody. Anti-Rabbit antibody488 (A-21206, Invitrogen, USA), and anti-Rabbit antibody594 (A-21207, Invitrogen, USA) as secondary antibodies were incubated at room temperature for 2 h. Cell nuclei were counterstained with DAPI. DAB (TA-060-QHDX Ther-moFisher Scientific, USA) was used for color development followed by hematoxylin counterstaining in immunohistochemistry assay. Three mice/six lenses from each group were evaluated to compare these proteins expression of these two groups and repeated 3 times.
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