#IGOR PRO PEAK LABEL SOFTWARE#

#IGOR PRO PEAK LABEL SOFTWARE#

The following parameters worked well for a Scientifica microscope and Micromanager software and gave an effective time resolution of 2.85 s. The main limitation on time resolution of image acquisition is the refocusing and movement of the XY translational stage. Adjustment of specific imaging parameters may be required for your application. Since the frame-rates of most cameras greatly exceed the duration of these calcium events, some time resolution can be sacrificed in order to gain additional image planes. Note: Calcium events in acute slice preparation can last several seconds at 20вЊвЂ“24вЊ, depending on the specific GCaMP variant that is utilized (fast or slow) and the cell type. Adjust the number of cycles to last approximately 10В min. Set up an imaging session that acquires data at two different XY stage positions and extends for several different depths into the slice. (2018) for additional tips on preparing slices from aging animals. Tissue from aged animals may be less healthy, but this is not limiting. Brain slices from juvenile animals are typically healthier and offer good expression of GCaMP indicator. Slice in low light conditions if possible, and ensure that sectioned tissue is shielded from room illumination by covering water-baths and slice-storage chambers with aluminum foil during recovery. Application of 70% ethanol may be used to keep bacteria from growing on tools or slice chambers.Д¬RITICAL: GCaMP indicators are light sensitive and will bleach if exposed to intense light. Thorough rinsing in water and scrubbing with reserved glassware brush will suffice. It is recommended not to use detergents or use department dishwashers. Reserve glassware, dissection tools, and vibratome for вЂњlive tissue only.вЂќ Do not share equipment with lab personnel that use fixative or other harsh chemicals, as it will seriously compromise your tissue. Continue shielding from light with sheets of aluminum foil.Д¬RITICAL: All materials and equipment involved in the preparation of acute brain slices must not have come in contact with fixative (paraformaldehyde or equivalent). Take care that bubbling is not so vigorous as to cause turbulence/tumbling of the sections.

Continue bubbling at 20вЊвЂ“24вЊ for 1вЂ“1.5 h. Ensure that sections are submerged and loosely separated. Validate that the virus level is decreasing during the injection by observing movement of a small bubble at the interface of oil and virus.Д¬hemicals, peptides, and recombinant proteinsД¬arefully discard the NMDG solution, and replace with 20вЊвЂ“24вЊ, bubbled HEPES holding solution. Care must be taken to prevent clogging at the tip. We have found, however, that appropriately small micropipettes induce less injury along the needle track due to the reduced diameter. Expression of hM3Dq can be confirmed by presence red fluorescence due to the mCherry reporter.ДЄlternatives: An alternative to micropipette injection is to use an injection needle made for a Hamilton syringe (ex: 33GA). GCaMP expression can be detected in fixed tissue using standard antibody staining against GFP if necessary. It is suggested to try a variety of dilutions to find the concentrations that work best. Occasionally combining two viruses has unexpected effects, and the ratios of the two must be adjusted to get desired expression levels. Close the wound, allow animal to recover, and monitor postoperatively according to institutional requirements.Д¬RITICAL: Confirm accuracy of viral injections.

Igor pro peak label