Immunohistochemistry of p38 in Mouse Retina

Mouse retina cryosections (10 μm) were fixed in 4% paraformaldehyde and blocked with 10% normal goat serum. The sections were rinsed with PBS, permeabilized with 0.1% Triton X-100, and incubated over night with anti-p38 antibody (rabbit polyclonal, ab7952, Abcam; Cambridge, MA). The slides were rinsed with PBS, and incubated with the anti-rabbit-FITC conjugated secondary antibody (NL006; R&D systems; Minneapolis, MN) for 1 hour [26 (link)]. After rinsing the slides with PBS, the sections were mounted with DAPI-containing mounting media (Vector Laboratories; Burlingame, CA), and imaged with an Olympus BX-UCB fluorescent microscope (20X magnification). The fluorescence intensity was quantified by using ImageJ software (version 1.44; NIH). Cryosections processed under similar conditions, except being incubated with the primary antibody, served as controls.

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Veluthakal R., Kumar B., Mohammad G., Kowluru A, & Kowluru R.A. (2015). Tiam1-Rac1 Axis Promotes Activation of p38 MAP Kinase in the Development of Diabetic Retinopathy: Evidence for a Requisite Role for Protein Palmitoylation. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 36(1), 208-220.

Publication 2015

ab7952 NL006 DAPI-containing mounting media BX-UCB fluorescent microscope

Anti antibody Antibody Cryosections Dapi Fitc Fluorescence Goat Microscope Mouse Paraformaldehyde Rabbit Retina Serum Triton x 100 Vector

Corresponding Organization :

Other organizations : Ophthalmology Associates (United States), Wayne State University, John D. Dingell VA Medical Center

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Variable analysis

independent variables

Tissue processing: 4% paraformaldehyde fixation, 10% normal goat serum blocking, 0.1% Triton X-100 permeabilization, and overnight incubation with anti-p38 antibody

dependent variables

Fluorescence intensity quantification using ImageJ software

control variables

Cryosection thickness (10 μm)
DAPI-containing mounting media
Olympus BX-UCB fluorescent microscope (20X magnification)

controls

Positive control: Cryosections processed under similar conditions, except being incubated with the primary antibody
Negative control: Not explicitly mentioned

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