Human and mouse eyelid sections were fixed with cold methanol for 15 minutes at −20°C. Following three phosphate-buffered saline (PBS) rinses for 5 minutes each, samples were incubated with 2% bovine serum albumin (BSA, Sigma-Aldrich Corp., St. Louis, MO) in PBS for 60 minutes. The slides were then placed overnight at 4°C in a moist chamber with antibodies specific for pimo (1 : 50; Hypoxyprobe Inc.), Glucose transporter-1 (Glut-1, 1:500, ab652, Abcam, Cambridge, MA), human CD31 (1:500, ab24590, Abcam), Notch1 (1: 10, bTAN20, Developmental Studies Hybridoma Bank, University of Iowa), or cytokeratin 14 (CK14, 1:500, ab181595, Abcam). After 3 additional PBS rinses, donkey anti-rabbit (1:200, ab150075, Abcam,), donkey anti-mouse (1:200, 2492098, EMD Millipore, Temecula, CA), or goat anti-rat (1: 200, a10522, Thermo Fisher Scientific, Grand Island, NY) secondary antibodies were applied the sections for 1 hour at room temperature. The primary antibodies were replaced with mouse or rabbit IgG as negative controls.
A10522
Manufactured by Thermo Fisher Scientific
The A10522 is a laboratory centrifuge designed for general-purpose applications. It provides a controlled environment for separating components of a liquid mixture based on their density differences. The centrifuge can accommodate a variety of sample volumes and rotor types to suit different laboratory needs.
Automatically generated - may contain errors
Lab products found in correlation
Ab652, by Abcam (1 mentions)
Ab181595, by Abcam (1 mentions)
Ab150075, by Abcam (1 mentions)
Ab24590, by Abcam (1 mentions)
Btan20, by Developmental Studies Hybridoma Bank (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
A21236, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dilactate dapi, by Merck Group (1 mentions)
A 21245, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Avantor (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Ab5694, by Abcam (1 mentions)
Ab13970, by Abcam (1 mentions)
Urea, by Chem-Supply (1 mentions)
Sucrose, by Chem-Supply (1 mentions)
Ab90810, by Abcam (1 mentions)
Ab5103, by Abcam (1 mentions)
Goat anti rat cy3, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
Eclipse ti, by Nikon (1 mentions)
4 protocols using a10522
1
Immunohistochemical Analysis of Eyelid Tissues
2
Immunohistochemistry Protocol for Tissue
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Neutral buffered formalin (NBF), Quadrol®, triethanolamine and 4′,6-diamidino-2-phenylindole (DAPI) dilactate were purchased from Sigma Aldrich. Normal goat serum was purchased from ThermoFisher. Urea and sucrose were purchased from Chem-Supply. Triton-X-100 was purchased from VWR International. The following primary antibodies were used for immunostaining: chicken anti-GFP (Abcam, ab13970, batch #s GR3190550-3 and -12), rat anti-F4/80 (Novus, NB600-404), rat anti-keratin 8 (DSHB, TROMA-I, batch #s 7/7/16 and 30/3/17), rabbit anti-keratin 5 (BioLegend, 905504, batch # B230397) and rabbit anti-SMA (Abcam, ab5694, batch # GR3183259-26). The following secondary antibodies were used: goat anti-chicken Alexa Fluor-488 (ThermoFisher, A21236), goat anti-rat Cy3 (ThermoFisher, A10522) and goat anti-rabbit Alexa Fluor-647 (ThermoFisher, A21245).
3
Visualizing Neutrophil Extracellular Traps
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After sectioning to 4 µm, the joint section was blocked with 10% normal goat serum for 45 minutes. The sections were incubated with an anti‐citrullinated Histone H3 antibody (Rabbit, 1:250, citrulline R2 + R8 + R17, ab5103; Abcam) and anti‐Ly6G antibody (1:300) or anti‐myeloperoxidase (MPO) (mouse, 2D4, 1:50, ab90810; Abcam). Isotype control antibodies were used. After three washes, Alexa 488‐goat anti‐rabbit (A‐11034; Invitrogen) to recognize the anti‐Histone H3 and Cy3‐goat anti‐rat (A10522; Invitrogen) to recognize the Ly6G were both incubated for 1 hour at room temperature. A 4',6‐diamidino‐2‐phenylindole (DAPI) nuclear stain was applied for 10 minutes followed by three washes of phosphate buffer saline (PBS) to remove residual dye. Slides were analyzed and imaged with a confocal microscope (Nikon Eclipse Ti). NETs or neutrophil initiated NETosis were identified according to the colocalization of DAPI, Ly6G, and NETs marker cit‐H3.
4
Antibody Immunofluorescence Staining Protocol
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Rabbit polyclonal antibodies against L. monocytogenes were a gift from Dr. Pascale Cossart (Institut Pasteur), mouse monoclonal antibodies against GFP were from Invitrogen (A-11120); antibodies against mouse p62-Ick ligand were from BD Biosciences (610832), rat polyclonal antibodies against LAMP1 were from Developmental Studies Hybridoma (1D4B) and antibodies against mono- and poly-ubiquitinated protein were from Biomol International (FK2; BML-PW8810-0500). All fluorescent secondary antibodies—goat anti rabbit 405, goat anti rat Cy3, goat anti mouse 568, goat anti mouse 488—were AlexaFluor conjugates from Molecular Probes (A31556; A10522, A11004; A11029; Invitrogen).
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