Neurohistology Lab #6. Nissl staining of frozen sections.

Materials 1. 3 slides of brain sections 2. Staining dishes 3. Staining racks 4. Deionized H20 (dH2O) 5. Ethanol (variety of concentrations) 6. Xylene 7. 0.1% cresyl violet 8. Mounting media 9. Coverslips

Purpose 1. Learn to perform the Nissl stain 2. Learn how to coverslip Background 1. As we discussed in class some weeks back, the Nissl stain labels rRNA (free and in the rER), which is abundant in neurons. The large amount of rER in neurons stain dark blue, appearing as large granules within neuronal cytoplasm; termed Nissl bodies. Nissl bodies can be using visualized with an aniline stain (cresyl violet) to label extranuclear RNA granules 2. As we discussed in class last class, the process of staining tissue is critical to the success of the stain. Adequate fixation, dehydration/rehydration, time in stain as well as coverslipping are all steps that can affect the success of staining. Introduction In this lab, our goal is to Nissl stain sections of cortex and to coverslip them so you can image them in the advanced imaging lab sometime next week (make appointments!). Sections have been cut for you using the vibratome, in addition to using your tissue from last week cut on the cryostate. You have been provided with 3 slides, each of which has coronal sections of cortex. Note these slides are not perfect and will have some artifact. We will stain each of the sections for different lengths of time in the cresyl violet (2 min, 5 min and 10 min) so you can directly assess the time-dependence of this stain. You will work in groups of 5 and each group will be responsible for one time point for the whole class. You need to make sure that you put one of your 3 slides in each of the staining racks. You can choose to put your cryostat sections in any of the racks. REMEBER TO LABEL YOUR SLIDES IN PENCIL.

Procedure Nissl Stain

1. dH2O #1 2. 70% EtOH #1 3. 100% EtOH #1 4. 100% EtOH #2 5. Xylene #1 6. Xylene #2 7. 100% EtOH #3 8. 100% EtOH #4 9. 100% EtOH #5 10. 70% EtOH #2 11. dH2O 12. dH2O 13. 0.1% cresyl violet 14. dH2O #2 15. 70% EtOH #2 16. 95% EtOH +Glacial Acetic Acid 17. 100% EtOH #1 18. 100% EtOH #2 19. Xylene #1 20. Xylene #2 2 min 10 dips (until stopped streaming) then 1 min 10 dips (until stopped streaming) then 1 min 1 min 1 min 1 min 10 dips (until stopped streaming) then 1 min 1 min 1 min 1 min 1 min 1 min 2, 5 or 10 min (you have 3 slides, one at each time point) few dips (10-15 sec) few dips (10-15 sec) 3 min 1 min 1 min 1 min 1 min (coverslip from here)

Note: these are all minimum times; leaving slides longer will not hurt them EXCEPT for differentiation step (#16) which is time dependent

Coverslipping
After completion of the staining process 1. Place a small drop of resin on a coverslip 2. Drain off the xylene being careful not to let the slide dry 3. Lower the slide (section side down) onto the resin at an angle to ensure no bubbles. 4. Quickly invert and place on slide tray overnight in hood . Note: Coverslip UNDER the hood.

Photographing slides
Slides will be dry by Friday. Make an appointment to go and photograph your slides or you can grab some images in class next week. Take images of different times in cresyl violet if possible in similar regions for better comparison. In your report dont forget to add labels, and label any ar tifacts. I am extending the deadline for the lab reports to 4/4/14 so everyone will be able to take pictures