Publication protocol
Triple-immunohistochemistry (IHC) was performed on experimental and control corneal sections to study expression of GFP, CD34 and CD45, or GFP, vimentin and alpha-smooth muscle actin (αSMA) at each time point. Corneal sections were prepared from all animals in each time point. The grouping of antigens in multiplex IHC was dictated by available primary antibodies. Briefly, slides containing three or four sections of each eye were washed twice in 1xPBS for five minutes and then fixed using 4% paraformaldehyde for only five minutes. Slides were then washed twice in 1X PBS for 5 minutes and sections were blocked using 5% normal donkey serum (#017000121, Jackson Laboratories, ME, USA) in 1X PBS for 30 minutes at room temperature. Primaries antibodies diluted in 5% normal donkey serum in 1X PBS, in the combinations described above, were placed on the slides and incubated at room temperature for one hour. Dilutions of primary antibodies were as follows: chicken anti-GFP antibody (ab13870, Abcam, Cambridge, UK) was diluted 1:2000, rat anti-CD45 antibody (MCD455, Thermo Fisher, MA, USA), sheep anti-CD34 (AF6518, R&D Systems, MN, USA) and rabbit anti-αSMA (ab5694, Abcam) were diluted 1:100 and the goat anti-vimentin (sc-7557, Santa Cruz, TX, USA) was diluted 1:50. After primary antibody incubation, slides were washed twice in 1X PBS for 5 minutes and secondary antibodies, also diluted in 5% normal donkey serum in 1X PBS, were added to the sections for one hour at room temperature as follows: Combination 1: donkey anti-chicken Alexa fluor 488 (#703-545-155, Jackson Immuno Research, PA, USA) at 1:500 + donkey anti-sheep Alexa fluor 647 (A-21448, Thermo Fisher) at 1:200 + donkey anti-rat Alexa fluor 594 (A-21209, Thermo Fisher) at 1:500; used for triple staining GFP, CD34 and CD45. Combination 2: donkey anti-chicken Alexa fluor 488 at 1:500 + donkey anti-rabbit Alexa fluor 568 (A-10042, Thermo Fisher) at 1:200 + donkey anti-goat Alexa fluor 647 (A-21447, Thermo Fisher) 1:200; used for triple staining GFP, αSMA and vimentin. Slides were finally washed three times in 1X PBS, air dried and mounted with Vectashield mounting media containing DAPI (H-1200, Vector Laboratories, CA, USA) to allow visualization of all nuclei. Sections for negative controls were included with incubations with secondary antibodies alone in the same dilutions as described above. The sections were analyzed and photographed with a Leica DM5000 microscope (Leica, Buffalo Grove, IL) equipped with Q-imaging Retiga 4000RV (Surrey, BC, Canada) camera and Image-Pro software (MediaCybernetics, Inc. Bethesda, MD).
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Manufacturer protocol
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