Computational protocol: ATF3 Regulates the Expression of AChE During Stress

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Protocol publication

[…] Mice were kept over-night in darkness for complete dark adaptation. Preparations for ERG recording were done under dim red light. The pupils were fully dilated with cyclopentolate hydrochloride 1%, and topical anesthesia (Localin) was applied to the cornea to prevent any potential discomfort. A heating pad was used to maintain normal body temperature. The ERG was recorded with a corneal electrode, using a drop of methylcellulose (Cellospan) to maintain corneal hydration, and to ensure electrical contact. Reference and ground electrodes, made of stainless steel surgical needles, were inserted through the ear (reference electrode), and under the skin in the back (ground electrode).Light stimuli were obtained from a Ganzfeld light source (LKC Technologies, Gaithersburg, MD, USA) with a maximum strength of 5.76 cd-s/m2. The strength of the light stimulus was attenuated manually by a set of neutral-density filters covering a range of 4 log units. Differential amplifiers (Grass Medical Instruments, Quincy, MA, USA) were used to amplify (10,000×) and filter (0.3–300 Hz) the ERG signals. The outputs of the amplifiers were digitized at a rate of 1 KHz by a computer equipped with a data acquisition board (LabVIEW 4.1 National Instruments, Austin, TX, USA). Six responses to six identical stimuli, applied at 10-s intervals, were averaged to improve signal/noise ratio. Data analysis was based on amplitude measurements of the ERG waves. The a-wave was measured from the baseline to its trough, and the b-wave was measured from the trough of the a-wave to the peak of the b-wave (e.g., Figure , inset). Since the ERG responses were severly reduced by the light exposure, we used amplitudes measured with the brightest test flash (5.76 cd-s/m2) to define the maximum response amplitude of the dark-adapted ERG a- and b-waves.ERG Vmax values were compared using ANOVA with Sigmaplot 11 software (Jandel Scientific, San Rafael, CA, USA). Data were considered statistically significant if p < 0.05. [...] Fixed eyes were washed in PBS, then cryo-protected in 15% and in 20% sucrose for 1 h each, and then in 30% sucrose for overnight at 4°C. The eyecups were embedded in OCT, cryo-cut into 16-μm thick sections along the vertical meridian, and mounted on glass slides. Retina cryo-sections were exposed to antibody against ATF3 (provided by Prof. Ami Aronheim, Technion), or against AChE (sc-6432, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200 in assay buffer; 0.1% Triton and 3% FBS in PBS, for overnight at 4°C. The sections were washed in PBS and incubated with secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), conjugated to Rhodamine (donkey anti-rabbit for ATF3) or Cy3 (donkey-anti goat for AChE) at a dilution of 1:500 in assay buffer; 0.1% Triton and 3% FBS in PBS, for 1 h at room temperature, while prevented from exposure to light. The sections were washed in PBS, stained with DAPI (1 mg/ml stock solution) at a 1:1000 dilution in PBS for 5 min, and then washed three times in PBS, mounted and cover-slipped for viewing by confocal microscopy (LSM-700, Zeiss). Omitting primary antibody in the immunostaining procedures served as a negative control.The sections were analyzed by 561 nm excitation wavelength, and later processed using IMARIS® scientific 3D/4D image processing and analysis software. For quantification, 2000–10,000 cells were counted in each tested field, and the fractions of cells stained for AChE or ATF3 were calculated. […]

Pipeline specifications

Software tools SigmaPlot, Imaris
Applications Miscellaneous, Laser scanning microscopy
Organisms Mus musculus