Tissues were fixed with 2 to 4% paraformaldehyde via transcardiac perfusion, after which dissected adipose pads were fixed overnight. The tissues were subsequently dehydrated through a series of ethanol washes and then embedded in paraffin for thin sectioning. For cross-sectional imaging of the inguinal adipose depots, a full-thickness section of the mouse flank (including the skin and adipose pad) was fixed and embedded in paraffin and then rotated and sectioned in the cross-sectional orientation. Immunohistochemistry analysis was performed by following heat antigen retrieval methods, and samples were stained with the following antibodies: anti–red fluorescent protein (RFP) (rabbit; 1:500; Rockland), anti-DPP4 (goat; 1:250; R&D Systems), anti-Pref1 (rabbit; R&D Systems), anti-Anxa3 (rabbit; Biorbyt), and anti-Pi16 (rabbit; mybiosource).
Anti dpp4
Manufactured by R&D Systems
Anti-DPP4 is a laboratory reagent designed for use in research applications. It is a protein that inhibits the enzymatic activity of dipeptidyl peptidase-4 (DPP4), a serine protease involved in the regulation of various physiological processes. The core function of Anti-DPP4 is to serve as a tool for researchers to study the role of DPP4 in biological systems.
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Lab products found in correlation
Anti red fluorescent protein, by Rockland Immunochemicals (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Tcs spe confocal microscope, by Leica camera (1 mentions)
Anti acetylated α tubulin, by Cell Signaling Technology (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Anti lamin b1, by BioLegend (1 mentions)
Turbo transfer, by Bio-Rad (1 mentions)
Precast sds page gel, by Bio-Rad (1 mentions)
Anti gapdh, by BioLegend (1 mentions)
Anti flag, by Merck Group (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Anti pdgfrα, by R&D Systems (1 mentions)
Anti ucp1, by Abcam (1 mentions)
Affinipure fab fragment, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 594 conjugated goat antibody to rabbit igg, by Thermo Fisher Scientific (1 mentions)
Click it plus edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti dpp4
1
Immunohistochemical Analysis of Adipose Tissue
2
Immunostaining of MERS-CoV Infection
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Antibodies and reagents used for immunostaining are listed here: Anti-acetylated α-tubulin (Cell Signaling, cat#5335S), anti-DPP4 (R&D Systems, cat#AF1180), anti-MERS-CoV N protein (Sino Biological, cat#40068-MM10), anti-cleaved caspase-3 (Cell Signaling, cat#9664), and Alexa Fluor 647 phalloidin (ThermoFisher Scientific, cat#A22287). Methods for immunolocalization of DPP4 in human airway epithelia were adapted from previous studies (7 (link)). Briefly, airway epithelia or cytospun BAL samples were fixed in 4% paraformaldehyde and permeabilized with 0.2 % Triton X-100. Following incubation with SuperBlock™ buffer, primary antibodies were applied to the fixed cells on slides, or to the apical and basolateral cell surfaces of epithelial sheets overnight, followed by washing with SuperBlock™ buffer three times. After a 1 h incubation with the appropriate Alexa Fluor secondary antibodies, followed by more washes, coverslips were mounted using Vectashield antifade mounting medium with DAPI (Vector Laboratories). Photomicrographs were taken using a Leica TCS SPE confocal microscope. The number and types of infected cells were counted manually using ImageJ.
3
Immunoblotting Analysis of ACE2, DYRK1A, and DPP4
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Around 1 × 106 cells were collected and lysed in Alfa Aesar Nonidet 40 (NP-40; 20 mM Tris–Hcl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM PMSF). Cell lysates were fractionated on SDS-PAGE pre-cast gels (BioRad) and transferred to a PVDF membrane by TurboTransfer (BioRad). Immunoblotting assays were performed with the following primary antibodies (1:1,000): anti-ACE2 (ProSci, cat#3217), anti-DYRK1A (Abcam, cat#ab259869), anti-FLAG (Sigma-Aldrich, cat#F3165), anti-DPP4 (R&D, cat#AF1180), anti-GAPDH (BioLegend, cat#649202), and anti-Lamin B1 (BioLegend, cat# 869801). Proteins were visualized with goat anti-mouse or goat anti-rabbit IgG secondary antibodies (1:5,000) diluted in 2% Omniblot milk (AmericanBio) in 1× TBST using a chemiluminescence detection system (BioRad ChemiDoc MP).
4
Multimodal Immunohistochemistry and Fluorescence Imaging
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Mouse tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin according to standard protocols. Immunohistochemical staining of paraffin sections was carried out with 1:200 anti-UCP1 (Abcam). For Immunofluorescence, slides were fixed with 4% PFA and permeabilized in ice cold methanol. Heat-mediated antigen retrieval with a 0.01 M citric acid (pH 6.0) was performed for 5 min in a microwave. After blocking with 4% BSA, the sections were blocked with unconjugated AffiniPure Fab Fragment (1:100; Jackson), then incubated with primary antibodies anti-UCP1 (rabbit; 1:200; Abcam), anti-red fluorescent protein (rabbit; 1:500; Rockland), anti-PDGFRα (goat; 1:200; R&D) or anti-DPP4 (goat; 1:200; R&D) followed by detection with secondary antibodies Alexa Fluor® 594 conjugated goat antibody to rabbit IgG (1:1,000; Invitrogen) or Alexa Fluor® 647 conjugated donkey antibody to goat IgG (1:1,000; Invitrogen). Finally, the sections were incubated with DAPI or Hoechst. The adipocytes were stained with BODIPY® 493/503 (Invitrogen). EdU incorporation assay was performed following the instruction of Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Invitrogen).
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