hRPE were fixed in 4% PFA for 10 min at room temperature, followed by three 10 min washes with PBS for 5 min. Cells were then washed in PBS−/− (Gibco) with 0.3% Triton™ X-100 (wash buffer) for five minutes, thrice, before blocking in 1% BSA, 3% FBS, 0.3% Triton X-100 in PBS−/− at room temperature for 20 min. Blocking buffer was then replaced with anti-ZO1 antibody (1:100, Invitrogen, cat. 40–2200) in blocking buffer in 4˚C overnight. On the following day, wash buffer was replaced three times every five minutes before the appropriate secondary antibody (anti-rabbit IgG H&L Alexa Fluor®647, 1:400, Invitrogen, cat. A-31573) blocking buffer was applied for 1 h at room temperature. After three more washes, slides were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories). Confocal images were acquired with Carl Zeiss LSM 780.
Anti rabbit igg h l alexa fluor 647
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Anti-rabbit IgG H&L Alexa Fluor®647 is a secondary antibody conjugated with the Alexa Fluor®647 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various applications such as immunohistochemistry, Western blotting, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (1 mentions)
Anti zo 1 antibody, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Anti mouse igg h l alexafluor488, by Thermo Fisher Scientific (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Anti neun, by Merck Group (1 mentions)
Fluoview fv10i, by Olympus (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Ma1 40220, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti rabbit igg h l alexa fluor 647
1
Immunofluorescence Imaging of Tight Junctions in hRPE
2
Immunostaining of Brain Tissue
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The PFA‐fixed slices were cut off from the membrane and transferred into 3% BSA solution for blocking. Afterward, the slices were incubated with primary antibodies (anti‐NeuN, Merck Millipore, Darmstadt, Germany and anti‐Iba1, Wako Chemicals GmbH, Neuss, Germany) overnight at 4°C. After the tissues were washed three times for 20 minutes, fluorescently labeled secondary antibodies (all Invitrogen, Karlsruhe, Germany; anti‐mouse IgG (H+L) AlexaFluor488, anti‐mouse IgG (H+L) AlexaFluor 546, anti‐rabbit IgG (H+L) AlexaFluor647, and anti‐rabbit IgG (H+L) AlexaFluor546, all 1:1000) were applied, and the slices were incubated for 2 h at room temperature. Again, after the slices were washed three times for 20 minutes each, the slices were transferred onto object slides and covered with fluorescence mounting medium (Dako, Santa Clara, USA), and a coverslip was placed on top.
3
Neutrophil Activation and Elastase Detection
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2 × 106 neutrophils were activated with 100 nM PMA with or without 500 µg/mL fluorescent AAT for 4 h at 37 °C. Afterwards, neutrophils were washed with PBS and fixed with 4% of paraformaldehyde for 10 min. After three washes with PBS, cells were treated with blocking solution (1% BSA in PBS) for 1 h at room temperature. Cells were then stained with anti-HNE mAb (dilution 1:50—MA1-40220—Invitrogen) in 0.5% BSA in PBS for 1 h at room temperature. After three washes in PBS, secondary mAbs (anti-rabbit IgG H&L, Alexa Fluor® 647—Invitrogen) were added in 0.5% BSA in PBS for 1 h at room temperature. Coverslips were then mounted using ProLong™ Gold Antifade Mountant with DAPI (Invitrogen) and analysed with confocal microscopy (Fluoview FV10i, Olympus, Tokyo, Japan).
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