Fresh tissue was fixed in 4% paraformaldehyde and embedded in paraffin. Five-micron sections were obtained, deparaffinised, and rehydrated as previously described. After antigen retrieval, endogenous peroxidase was blocked using 3% hydrogen peroxide at room temperature for 10 min. Sections were blocked with 5% BSA and then incubated with primary antibody (11β-HSD1, 1:100, Abcam, Cambridge, United Kingdom) in a humid chamber at 4 °C overnight, followed by incubation with an HRP-conjugated secondary antibody (1:200) at room temperature for 1 h. After colour development through incubation with diaminobenzidine, the sections were counterstained with hematoxylin. The developed tissue sections were visualised under a microscope (Olympus Corporation).
11β hsd1
Manufactured by Abcam
Sourced in United Kingdom, United States
11β-HSD1 is an enzyme that catalyzes the interconversion of physiologically active glucocorticoids and their inactive 11-keto forms. It plays a role in regulating glucocorticoid action in target tissues.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Pro prep, by iNtRON Biotechnology (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
11β hsd2, by Santa Cruz Biotechnology (1 mentions)
Dako pd l1 22c3 pharmdx kit, by Agilent Technologies (1 mentions)
Histofine kit, by Nichirei Biosciences (1 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
C57bl 6 mice, by Charles River Laboratories (1 mentions)
Cox 2, by GeneTex (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg, by GE Healthcare (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Image studio software, by LI COR (1 mentions)
C ebpα, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
8 protocols using 11β hsd1
1
Immunohistochemical Analysis of 11β-HSD1
2
Evaluating Antioxidant Effects of A. spectabilis
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The biological
activities of samples isolated from the Korean endemic plant A. spectabilis were evaluated as described in our
previous study with minor modifications.27 (link) HaCaT cells (Korean Cell Line Bank, Seoul, Republic of Korea) were
seeded into 60 mm plates (1 × 106 cells/well) in Dulbecco’s
modified Eagle medium including 5% fetal bovine serum and 1% penicillin
and streptomycin. Samples at a 2.5 μM concentration were treated
in the wells for 2 h and then exposed to UVB at 125 mJ/cm2 using a UV lamp. After UVB irradiation, the cells were harvested,
washed with cold PBS (1×), and lysed in PRO-PREP (iNtRON Biotechnology,
Inc., Sungnam, Republic of Korea). Equal amounts of protein (30 μg)
obtained from the lysed cells were loaded onto 10% SDS gels and then
transferred to PVDF membranes (Millipore, Billerica, MA, USA). The
transferred membranes were incubated with primary antibodies (1:1000)
including GRs (Cell Signaling, MA, USA), 11β-HSD1 (Abcam, Cambridge,
UK), and α-tubulin (Cell Signaling). After 1 day, the attached
primary antibody membranes were incubated with secondary antibodies
(1:2000) for 2 h. All membranes were analyzed using a ChemiDoc XRS+
imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The
detected band intensity was calculated using Bio-Rad Quantity One
software (ver. 4.3.0, Bio-Rad).
activities of samples isolated from the Korean endemic plant A. spectabilis were evaluated as described in our
previous study with minor modifications.27 (link) HaCaT cells (Korean Cell Line Bank, Seoul, Republic of Korea) were
seeded into 60 mm plates (1 × 106 cells/well) in Dulbecco’s
modified Eagle medium including 5% fetal bovine serum and 1% penicillin
and streptomycin. Samples at a 2.5 μM concentration were treated
in the wells for 2 h and then exposed to UVB at 125 mJ/cm2 using a UV lamp. After UVB irradiation, the cells were harvested,
washed with cold PBS (1×), and lysed in PRO-PREP (iNtRON Biotechnology,
Inc., Sungnam, Republic of Korea). Equal amounts of protein (30 μg)
obtained from the lysed cells were loaded onto 10% SDS gels and then
transferred to PVDF membranes (Millipore, Billerica, MA, USA). The
transferred membranes were incubated with primary antibodies (1:1000)
including GRs (Cell Signaling, MA, USA), 11β-HSD1 (Abcam, Cambridge,
UK), and α-tubulin (Cell Signaling). After 1 day, the attached
primary antibody membranes were incubated with secondary antibodies
(1:2000) for 2 h. All membranes were analyzed using a ChemiDoc XRS+
imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The
detected band intensity was calculated using Bio-Rad Quantity One
software (ver. 4.3.0, Bio-Rad).
3
Immunohistochemical Antibody Characterization
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The characteristics of the antibodies used were as follows: 11βHSD1 ([Abcam, Cambridge, UK], EP9406(2), Rabbit monoclonal, 1:200, Autoclave in citrate buffer), 11βHSD2 ([Santa Cruz Biotechnology, Santa Cruz, USA], C-9, Mouse monoclonal, 1:200), GR ([Cell Signaling Technology, Danvers, MA, USA], D6H2L, Rabbit monoclonal, 1:400, Autoclave in citrate buffer), CD3 ([Dako, Glostrup, Denmark], F7.2.38, Mouse monoclonal, 1:500, Autoclave in citrate buffer) and CD8 ([Dako, Glostrup, Denmark], C8/144B, Mouse monoclonal, 1:50, Autoclave in citrate buffer). Immunohistochemistry for PD-L1 was performed using the Dako PD-L1 22C3 pharmDx kit (Dako, Carpinteria, CA) on the Dako Link 48 platform. A Histofine Kit (Nichirei, Tokyo, Japan) using the streptavidin–biotin amplification method was used for 11βHSD1, GR, CD3 and CD8, and the EnVision kit (Dako, Agilent Technologies, Inc., Santa Clara, CA, USA) was used for 11βHSD2 in this examination. The antigen–antibody complex was visualised using the 3,3ʹ-diaminobenzidine (DAB) solution (1 mM DAB, 50 mM Tris-HCL buffer, pH 7.6 and 0.006% H2O2) and counterstained with haematoxylin.
4
Gestational Changes in Fetal Membrane Proteins
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Animal experimentation was conducted according to approved standards for animal care by the Institutional Review Board of Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine. C57BL/6 mice (Charles River, Beijing, China) aging from 10 to 13 weeks were mated overnight. The presence of a vaginal plug in the morning was counted as gestational day 0.5. Fetal membranes were collected on gestational days 14.5 and 18.5, and fixed for the study of the distribution of C/EBPδ, COX‐2, and 11β‐HSD1 with immunohistochemical staining of paraffin‐embedded tissue sections using antibodies against C/EBPδ (GeneTex), COX‐2 (Cell Signaling Technology), and 11β‐HSD1 (Abcam). To study gestational changes of C/EBPδ, COX‐2, and 11β‐HSD1, fetal membranes were collected on gestational days 14.5, 15.5, 16.5, 17.5, and 18.5 for protein extraction and measurements of C/EBPδ, COX‐2, and 11β‐HSD1 abundance with Western blotting.
5
Protein Expression Analysis in Tissue Extracts
6
Quantitative Analysis of Hepatic Protein Expression
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Whole liver was immediately frozen using clamps pre-cooled to the temperature of liquid nitrogen and stored at −80° for Western blot analysis. Briefly, samples were analyzed for protein content, and separated on 4%–12% Bis-Tris gels at 150 volts for 1.5 h. Transfer was for 2 h at 30 volts on ice. Blots were incubated with primary antibody to GR (1:1000; Cell Signaling Technology, Danvers, MA, USA), PEPCK (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), 11β-HSD1 (1:1000; Abcam Cambridge, MA, USA), G6Pase α (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and G6Pase β (1:200; Santa Cruz Biotechnology Inc.) overnight at 4°C. To probe for actin, blots were incubated with anti-actin primary antibody (1:5000; EMD Millipore, Billerica, MA, USA) for 1 h at room temperature. After washing, blots were incubated with secondary antibody, anti-rabbit Immunoglobulin G (IgG) (heavy and light chains) DyLight (1:100,000; Cell Signaling Technology) and simultaneously with anti-mouse IgG (H + L) DyLight (1:15,000; Cell Signaling Technology), for 1 h at room temperature. Images of membranes were obtained with the abundance of all proteins of interest normalized to actin, which was the internal control. Band density was analyzed using Odyssey-Clx (LI-COR, Lincoln, NE, USA) and Image Studio (LI-COR).
7
Hippocampal Protein Expression Analysis
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Four mice were decapitated under 1% pentobarbital sodium, and their brains were collected immediately. Fresh hippocampal tissue samples were a random subset picked blindly. Tissue and cells were homogenised in lysis buffer with protease inhibitor mixture and were centrifuged at 12,000×g for 15 min, and then the supernatants were collected. Proteins were separated by SDS-PAGE gel (10% separation gel) and transferred to a PVDF membrane (Merck & Co., Inc., Whitehouse Station, NJ, USA, Germany). The membrane was blocked with 5% fat-free milk solution for 1 h at room temperature and incubated with respective primary antibodies overnight at 4 °C: 11β-HSD1 (ab169785, 1:1000, Abcam, Cambridge, United Kingdom), BCL-2 (#3498, 1:1000; Cell Signaling Technology), BAX (#14796, 1:1000; Cell Signaling Technology), Cleaved CASPASE-3 (#9661, 1:1000; Cell Signaling Technology) and GAPDH (#5174, 1:1000; Cell Signaling Technology). After being washed with TBST three times, immunoreactive bands were incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (A0208, 1:2000; Beyotime) or goat anti-mouse secondary antibody (#SSA007, 1:5000, Sino biological). The ECL procedure (Bio-Rad) was used to detect the proteins.
8
Western Blot Analysis of Collagen and Autophagy Proteins
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After treatment, the cells were lysed in ice-cold RIPA lysis buffer (Active Motif) containing a protease inhibitor cocktail (Sigma). After determination of protein concentration, the extracted protein was analyzed with Western blotting following a standard protocol using primary antibodies against COL3A1 (1:1000, Novus, Littleton, CO, USA), COL1A1 (a major chain of collagen I, 1:1000, Novus), ATG7 (1:1000, Cell Signaling), GR (1:1000, Santa Cruz Biotechnology) and 11β-HSD1 (1:500, Abcam). Internal loading controls were probed with antibodies against GAPDH (1:5000, Proteintech, Rosemont, IL, USA) or β-Actin (1:10000, Proteintech). The bands were visualized using a G-Box iChemi Chemiluminescence image capture system (Syngene, Cambridge, UK). The ratio of band intensities of COL3A1, COL1A1, ATG7, GR and 11β-HSD1 over GAPDH or β-Actin was obtained, respectively, as a measure of target protein abundance.
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