All tissue samples were fixed and processed into paraffin blocks. After dewaxing, antigen repair (10 mmol/L sodium citrate for 5 min in a pressure cooker) and removing endogenous enzymes in Hydrogen Peroxide Block, the sections were incubated with the primary antibodies anti-GFP (ab13970, Abcam, UK, the dilution ratio was 1:500) and anti-Perilipin (ab3526, Abcam, UK, the dilution ratio was 1:200) at 4ºC overnight. Then the samples were incubated in secondary antibody (703-545-155, Jackson ImmunoResearch, USA; ab150074, Abcam, UK, the dilution ratio was 1:500) for 1-2 h at room temperature in the dark. After rinsing three times in 1X PBS for 5 min each, the slides were counterstained with Hoechst (C1026, Beyotime, China, the dilution ratio was 1:500) to reveal cell nuclei. Finally, fluorescence microscopy was used to evaluate specimen.
Ab3526
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab3526 is an antibody that can be used for protein detection and analysis. It is a highly specific and sensitive tool for researchers in the life sciences field.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Ab13970, by Abcam (3 mentions)
Ab9256, by Abcam (3 mentions)
Prolong gold antifade reagent with dapi, by Cell Signaling Technology (3 mentions)
Ab150074, by Abcam (2 mentions)
Ab10983, by Abcam (2 mentions)
Ab150080, by Abcam (2 mentions)
Fv1000, by Olympus (2 mentions)
Immunofluorescence application solutions kit, by Cell Signaling Technology (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab77115, by Abcam (1 mentions)
Ab2739, by Abcam (1 mentions)
Ab124807, by Abcam (1 mentions)
Ab3340, by Abcam (1 mentions)
Ab99532, by Abcam (1 mentions)
Ab96718, by Abcam (1 mentions)
β actin sc 8432, by Santa Cruz Biotechnology (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Ha h3663, by Merck Group (1 mentions)
32 protocols using ab3526
1
Immunofluorescence Staining of Paraffin Sections
2
Paraffin-embedded Immunofluorescence Staining
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All tissue samples were fixed and processed into paraffin blocks. After dewaxing, antigen repair (10 mmol l−1 sodium citrate for 5 min in a pressure cooker) and removing endogenous enzymes in hydrogen peroxide block, the sections were incubated with the primary antibodies anti-GFP (ab13970, Abcam, UK, the dilution ratio was 1:500) and anti-Perilipin (ab3526, Abcam, UK, the dilution ratio was 1:200) at 4 °C overnight. Then the samples were incubated in secondary antibody (703–545–155, Jackson ImmunoResearch, USA; ab150074, Abcam, UK, the dilution ratio was 1:500) for 2 h at room temperature in the dark. After rinsing three times in 1× PBS for 5 min each, the slides were counterstained with Hoechst (C1026, Beyotime, China, the dilution ratio was 1:500) to reveal cell nuclei. Finally, fluorescence microscopy was used to evaluate specimen.
3
Adipocyte Lipid Metabolism Regulation
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Antibodies against OGT (ab96718), OGA (MGEA5, ab124807), O-GlcNAc (RL2, ab2739), PLIN1 (ab3526), SNAP23 (ab3340), ATGL (ab99532), DGAT1 (11561-1-AP), Fsp27 (CIDE C, ab77115), and p-Ser (phosphoserine, PSR-45) (Abcam); p492-phosphorylated PLIN1 (4855) and p517-phosphorylated PLIN1 (4856) (Vala Sciences); HA (H3663) (Sigma-Aldrich); CGI-58 (ABHD5, 12201-1-AP), PLIN2 (15294-1-AP), and PLIN3 (TIP47, 10694-1-AP) (Proteintech); p563-HSL (4139), phospho-Akt (Ser473, 9271), and Akt (9272) (Cell Signaling Technology); Myc (sc-40), DGAT2 (sc-66859), and β-actin (sc-8432) (Santa Cruz Biotechnology) were purchased from the indicated sources. Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology. Alexa Fluor 594-conjugated secondary antibodies, BODIPY 493/503, and BODIPY 558/568 C12 fatty acid were obtained from Thermo Fisher Scientific. 4′,6-Diamidino-2-phenylindole (DAPI), OA, Fsk, and IBMX were from Sigma-Aldrich. CL-316,243 was from R&D Systems. OGT inhibitor ST045849 (ST04) was purchased from TimTec. TMG was from Cayman Chemical.
4
Antibody Sources for Plin1 and Plin2
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Polyclonal antibodies against Plin1 or Plin2 [7 (link), 8 (link), 10 (link), 16 (link)] were gifts from the laboratory of C. Londos (US National Institutes of Health). This Plin1 antibody was used for immunostaining and another anti-Plin1 antiserum from Abcam (#ab3526) was used for immunoblotting. The sources of other antibodies used were listed as follows: rabbit anti-ATGL from Cayman Chemical (Cat# 10006409), rabbit anti-HSL from Cell Signaling Technology (Cat# 4107s), rat anti-CD36 from (R&D, MAB1955), mouse anti-ABCA1 (Abcam, ab18180), and mouse monoclonal anti-CD68 (Abcam, #ab955),
5
Comprehensive Protein Expression Analysis
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Calnexin, VEGFA, CD31, HIF-1 alpha, (AB0037, AB0063, AB0092, AB0112, Sicgen, Portugal), β-actin, α-tubulin (A5316, T6199, Sigma, USA), PPARγ, AKT, p-AKT(Ser473), VEGFR2, PI3K (#2443, #9272, #4058, #2479, #4249, Cell Signaling, USA), ANG-2, F4/80, Perilipin-A, GLUT4, p-IR(Y1361), GLO-I, C/EBPalpha, PGC1alpha, ANGPTL4, AT1, HIF-2 alpha (Ab8452, Ab74383, Ab3526, Ab65267, Ab60946, Ab96032, Ab40764, Ab191838, Ab2920, Ab9391, Ab8365, Abcam, UK), CD11c, CD206 (bs-2058R, bs-2664R Bioss, USA) TIE-2, IRβ (sc-324, sc-57342, SantaCruz Biotechnology, USA), CEL (KH025, TransGenic Inc, Japan).
6
Perilipin Immunohistochemistry in Skin Adipocytes
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Immunohistochemical detection of perilipin (1:200, ab3526, Abcam Cambridge, MA, USA) was performed on formalin-fixed samples that were processed, embedded in paraffin blocks and sectioned at 5 μm. Heat-induced antigen retrieval followed by ABC detection (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA, USA) was performed using the protocols described previously [11 (link)]. Peroxidase activity was revealed using 3.3-diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA) as a substrate. Slides were counterstained with hematoxylin and visualized using an Olympus microscope (BX43, Olympus, Tokyo, Japan).
Measurements of percentage of the adipocyte area in the skin, and diameter and size of intradermal adipocytes were performed using ImageJ software version v1.52a (National Institutes of Health, Bethesda, MD, USA) (number of mice n = 5 per group).
Measurements of percentage of the adipocyte area in the skin, and diameter and size of intradermal adipocytes were performed using ImageJ software version v1.52a (National Institutes of Health, Bethesda, MD, USA) (number of mice n = 5 per group).
7
Immunohistochemical Analysis of Adipose Tissue
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Antibodies included rabbit anti-UCP1 (ab10983, Abcam), rabbit anti-prohibitin (ab28172, Abcam), rabbit anti–perilipin A (ab3526, Abcam), rat anti–mouse endomucin (14-5851, eBioscience) and mouse anti–β-actin (TA346894, ZSGB-Bio). The HRP DAB detection system for rabbit or rat antibodies (ZLI-9018, ZSGB-Bio) was used for immunohistochemical staining. Rodent HFD with 60% kcal% fat was from Research Diets.
8
Adipogenesis and Angiogenesis in Implants
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After fixation in 4% paraformaldehyde for 24–48 h, samples were embedded in paraffin and sectioned. Hematoxylin and eosin (HE) were used to examine the morphology of the implants at each study time point. Immunohistochemical staining was performed for the lipogenic marker Perilipin-1 (Abcam ab3526, Cambridge, UK, 1:1000) and CD31 (Abcam ab28364, Cambridge, UK, 1:4000) was used to locate vascular endothelial cells. Sections were scanned using a microscopic digital slice scanning system (Motic EasyScan Pro 6). Image J software was used to calculate the area proportion of panoramic Perilipin-1-positive adipocytes to assess adipogenesis in implants. The CD31-positive area proportion of 5 random fields of view (×100 magnification) was calculated to assess angiogenesis in the implants in each group.
9
Quantitative Adipogenesis Analysis of Tissue Samples
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Tissue samples were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin for staining with hematoxylin and eosin (HE).
Tissue samples were immunohistochemically stained with rabbit anti-mouse histone H2AX phosphorylation (γH2A.X) (1:200, ab26350; Abcam, Cambridge, UK) and rabbit anti-mouse p21 (1:200, ab188224; Abcam) primary antibodies and then with a horseradish peroxidase-conjugated goat anti-rabbit IgG H&L secondary antibody (1:1000, ab205718; Abcam).
After grafting, tissue sample sections were immunofluorescently stained with a rabbit anti-mouse perilipin primary antibody (1:200, ab3526; Abcam), before being washed and labeled with an Alexa Fluor® 594-conjugated goat anti-rabbit IgG secondary antibody (1:1000, ab150080; Abcam). Nuclei were stained with DAPI (1:10000, D9542; Sigma, St. Louis, MO, USA). To quantitate adipogenesis, the percentage of perilipin-positive areas was determined by dividing the positive area by the total area. All images were captured with a microscope (Olympus BX63; Olympus, Tokyo, Japan). Images were analyzed using ImageJ software.
Tissue samples were immunohistochemically stained with rabbit anti-mouse histone H2AX phosphorylation (γH2A.X) (1:200, ab26350; Abcam, Cambridge, UK) and rabbit anti-mouse p21 (1:200, ab188224; Abcam) primary antibodies and then with a horseradish peroxidase-conjugated goat anti-rabbit IgG H&L secondary antibody (1:1000, ab205718; Abcam).
After grafting, tissue sample sections were immunofluorescently stained with a rabbit anti-mouse perilipin primary antibody (1:200, ab3526; Abcam), before being washed and labeled with an Alexa Fluor® 594-conjugated goat anti-rabbit IgG secondary antibody (1:1000, ab150080; Abcam). Nuclei were stained with DAPI (1:10000, D9542; Sigma, St. Louis, MO, USA). To quantitate adipogenesis, the percentage of perilipin-positive areas was determined by dividing the positive area by the total area. All images were captured with a microscope (Olympus BX63; Olympus, Tokyo, Japan). Images were analyzed using ImageJ software.
10
Immunocytochemical Analysis of Adipocyte Markers
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Cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. Samples were washed three times with 1× PBS and incubated in blocking/permeabilization buffer (5.0% goat serum, 2.0% bovine serum albumin, 0.5% Triton X-100 in PBS) overnight at 4°C. For PDGFRα staining, blocking/permeabilization buffer without the goat serum (2.0% bovine serum albumin, 0.5% Triton X-100 in PBS) was used. The following primary and secondary antibodies were used for immunocytochemistry in this study: anti-PLIN (Abcam; ab3526; 1:200); anti- αSMA (Abcam; ab7817; 1:200); anti-YAP (Santa Cruz Biotechnology; sc101199; 1:200); anti-TAZ (Cell Signaling Technology; 83669S; 1:100); anti-PPARγ (Santa Cruz Biotechnology; sc7273; 1:200); anti-PDGFRα (R&D Systems; AF1062; 1:200); anti-β-CATENIN (Cell Signaling Technology; 8480S; 1:100); goat anti-rabbit Alexa 488 (Thermo Fisher Scientific; A11008; 1:500); goat anti-mouse Alexa Fluor 546 (Thermo Fisher Scientific; A11003; 1:500); goat anti-mouse Alexa 488 (Thermo Fisher Scientific; PIA32723; 1:500); and goat anti-rabbit Alexa 647 (Thermo Fisher Scientific; PIA32733; 1:500). Hoechst 33342 (Thermo Fisher Scientific; 1:1,000) and Phalloidin-iFluor 488 (Cayman Chemicals; 1:1,000) were used to stain nuclei and F-actin, respectively.
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