Immunofluorescence (IF) experiments were performed using standard procedures. Seven-micrometer lung sections were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and treated with Super Block for blocking (Sytek Laboratories, West Logan, UT). Antibodies were diluted in buffered diluent (Immco Diagnostics, Buffalo, NY). Primary antibodies included either rabbit anti-HDAC1 1° (1:50, Abcam) or rabbit anti-phosphorylated-HDAC1 1° (1:200, Abcam) and donkey anti-rabbit Alexa-Fluor 594 2° antibody (1:500, Abcam). Slides were mounted with Prolong Gold with Dapi (Life Technologies, Carlsbad, CA). Pictures were taken at 20× to 40× using an Axiovert 200M fluorescence microscope (Zeiss, Oberkochen, Germany). n = 4 to 6 per group. Alveolar macrophages were isolated from bronchoalveolar (BAL) fluid and prepared for IF experiments, as described previously.26 (link) Standard IF procedures were followed, using 4% paraformaldehyde fixation, 0.2% Triton X-100 permeabilization, superblock blocking, anti-phosphorylated-HDAC1 (1:100, Abcam) 1° antibody, and donkey anti-rabbit AlexaFluor 594 (1:500, Abcam) 2° antibody. Slides were mounted with Prolong Gold with Dapi (Life Technologies). Pictures were taken at 100× using an Axiovert 200M fluorescence microscope (Zeiss) n = 5 to 7 per group.
Donkey anti rabbit alexafluor 594
Manufactured by Abcam
Donkey anti-rabbit AlexaFluor 594 is a secondary antibody conjugated with the AlexaFluor 594 fluorescent dye. It is designed to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions)
Axiovert 200m fluorescence microscope, by Zeiss (1 mentions)
Ab80892, by Abcam (1 mentions)
Donkey antimouse alexafluor 488, by Abcam (1 mentions)
Sc 5279, by Abcam (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
2 protocols using donkey anti rabbit alexafluor 594
1
Immunofluorescence Imaging of HDAC1 and Phospho-HDAC1
2
Immunofluorescence Staining of mESCs
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mESCs were fixed in 4% paraformaldehyde (PFA) for 20 minutes at room temperature (RT). Then the mESCs were washed three times for 5 minutes in PBS at RT and permeabilized in PBS + Triton-X for 8 minutes. After washing mESCs in PBS, they were kept in blocking solution (PBS + BSA + tween-20) for one hour at RT. mESCs were then incubated overnight in primary antibodies, Oct4 (1:200, Santa Cruz, SC-5279) and Nanog (1:100, Abcam, ab80892) at 4 °C. The following day, mESCs were washed and incubated in secondary antibodies, donkey anti rabbit alexa fluor 594 (Abcam, 1/500) and donkey anti mouse alexa fluor 488 (Abcam, 1/500) for one hour at RT. mESCs were washed further prior to mounting in Vectashield mounting medium with Dapi (Vector Laboratories) and visualized using a fluorescence microscope (Olympus 1 × 71, Olympus, Aartselaar, Belgium).
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