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[…] Transfected organotypic slice cultures (constructs indicated in the text) were pharmacologically treated (as indicated in the text), then subjected to photostimulation. 24 to 30 hr post-PPS, slice cultures were placed in a Warner chamber filled with warm Tyrode’s solution containing (in mM): 150 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, 10 HEPES. Single plane images (1024 × 1024 pixel resolution) were acquired using a Plan-Neofluar 63X/1.3 oil immersion objectives mounted on a Zeiss LSM 510 inverted confocal microscope. Quantification of green (MRE-GFP) and red (mCherry) somal fluorescence was performed using ImageJ software as previously described (; ). MRE-GFP expression profile was determined by normalizing GFP fluorescence intensity over background fluorescence intensity, and the resultant value was normalized to the average value from the control group without photostimulation. Background fluorescence was determined by a region equal to the region area of the analyzed neuron in the field of view adjacent to the neuron. mCherry fluorescence was not different between any group within studies, suggesting that pharmacological and/or genetic manipulations did not alter the expression of mCherry-tagged constructs (ChR2 or Cre). Two to three independent slice cultures (litters) were used in each imaging study. Statistical significance between groups was determined with a two-factor ANOVA (factor 1 = photostimulation, factor 2 = genotype or drug treatment) with Tukey’s multiple comparison test. [...] At 3 DIV, organotypic hippocampal slice culture were biolistically transfected with PA1-GFP, which expresses a myristoylated form of GFP to enhance filling of spines, and/or ChR2H134R-mCherry. At 8–13 d post-transfection (12–17 DIV), slices were subjected to photostimulation as indicated in the text. Slices were fixed in 2.5% PFA/4% sucrose for 1.5 hr, followed by permeabilization in 0.5% Triton X-100/10% normal donkey serum for 2 hr. Slices were incubated with 1° anti-GFP antibody (Aves Labs) at 4°C overnight, followed by incubation with 2° anti-chicken Alexa Fluor 488 antibody (Life Technologies) for 4 hr at room temperature. Secondary apical dendrites (150–200 μM from soma) of transfected CA1 neurons were imaged using a Zeiss LSM 780 2-photon laser scanning microscope. Images were obtained using an excitation wavelength of 920 nm and a 40 × 1.4 NA oil immersion objective. An interval of 0.3 μM and pixel resolution of 2048 × 2048 was used to acquire Z-stacks, generating images with pixel dimensions of 0.07 × 0.07 × 0.3 μM. For each neuron, 1–2 regions of interest were acquired. Images were analyzed using NeuronStudio, as previously described (; ; ). Imaging experiments were performed blind to treatment. […]

Pipeline specifications

Software tools ImageJ, NeuronStudio
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Chemicals alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid