vGAT Preview

Another experiment in posting 'Original Size' images...

Single channel image of Cy 3-labeled rb anti-vGAT (1:10k) in the LHA. Original is 5 x 4 tile mosaic of 1200 x1200 images taken at 20X. The full-size (5443 x 4387) image was sharpened, cropped, converted to sRGB, and saved as a JPEG for upload.

Volocity: Exporting Channels

In a nutshell, the secret to exporting individual channels is to

1. Turn off all other channels (One of the buttons in the sidebar on the right, available when viewing the acquired image)
2. Capture a screen shot (Under the'Image' Menu select 'Capture a Screenshot...') using the 'Current View Size' option
3. Export the screenshot as an OME TIFF
4. Repeat for each channel

Here's how it looks using a 2 channel image of CCK in green and NeuroTrace Far Red in the red channel. These images were minimally processed. They were exported from Volocity and taken into PS 5 via Lightroom. In PS they were assigned and converted to a color profile and auto contrast was applied. The image was then re-sized to 1024x1190, converted to sRGB and saved as a JPEG. The images below are 1) a snapshot of the Cy 2-rabbit anti-CCK in the green channel, 2) a snapshot of NeuroTrace FRed in the red channel, and 3) a snapshot of the composite Red + Green channel. Note that the NeuroTrace was color changed in Volocity. Exporting works the same for mixed colors, but exporting the individual channels becomes more important. This is because channels in Volocity refer to data regardless of color, whereas in PS and everywhere else channels refer to color without regard for content. In other words, most colors use more than one channel  (e.g., Purple = Red + Blue, Yellow = Red + Green, etc.) making it more difficult to make selective changes to the component labels.

1: Green Channel ( Cy 2-rabbit anti-CCK)

2: Red Channel ( NeuroTrace FRed)

3: Red and Green Channel Composite

Nissl staining protocol

Thionin Staining Protocol as of 2/5/13
Protocol based on the 2003 procedure (on the wall in the wet lab)

Part 1: de-lipidation
1. 70% ethyl alcohol (EtOH) 2 changes, 2 min each
2. 95% EtOH 2 solutions, 2 min each
3. 100% EtOH 4 solutions, 2 min each
4. Xylene 3 solutions, 15 min each (under hood)

Part 2: rehydrating
1. 100% EtOH 4 solutions, 2 min each
2. 95% EtOH 2 solutions, 2 min each
3. 70% EtOH 2 solutions, 2 min each
4. 50% EtOH 1 solution, 2 min
5. deionized H2O (dH2O) 1 solution, 2 min

Part 3: staining and differentiation
1. dip in thionin dye, 30 seconds
2. dip in dH2O, 30 seconds
3. 50% EtOH 1 solution, 2 min
4. 70% EtOH 2 solutions, 2 min each
5. 95% EtOH 2 solutions, 2 min each
6. If overstained, differentiate in 95% EtOH with glacial Acetic Acid (1.5 ml gAA/250 ml EtOH)*
7. 100% EtOH 4 solutions, 2 min each
8. Xylene 3 solutions, 5 min each
9. Coverslip and dry flat