8/12/2019 NHT Lab 6

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Neurohistology Lab #6.Nissl staining of frozen sections.

Materials

1. 3 slides of brain sections

2. Staining dishes3. Staining racks4. Deionized H 20 (dH 2O)5. Ethanol (variety of concentrations)6. Xylene7. 0.1% cresyl violet8. Mounting media9. Coverslips

Purpose

1. Learn to perform the Nissl stain2. Learn how to coverslip

Background1. 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 granuleswithin 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 canaffect 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 theadvanced 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 cuton the cryostate. You have been provided with 3 slides, each of which has coronal sections of cortex. Notethese slides are not perfect and will have some artifact. We will stain each of the sections for different lengthsof time in the cresyl violet (2 min, 5 min and 10 min) so you can directly assess the time-dependence of thisstain. 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 putyour cryostat sections in any of the racks. REMEBER TO LABEL YOUR SLIDES IN PENCIL.

8/12/2019 NHT Lab 6

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Procedure

Nissl Stain1. dH2O #1 2 min2. 70% EtOH #1 10 dips (until stopped streaming) then 1 min3. 100% EtOH #1 10 dips (until stopped streaming) then 1 min

4. 100% EtOH #2 1 min5. Xylene #1 1 min6. Xylene #2 1 min7. 100% EtOH #3 10 dips (until stopped streaming) then 1 min8. 100% EtOH #4 1 min9. 100% EtOH #5 1 min10. 70% EtOH #2 1 min11. dH2O 1 min12. dH2O 1 min

13. 0.1% cresyl violet 2, 5 or 10 min (you have 3 slides, one at each time point)14. dH2O #2 few dips (10-15 sec)15. 70% EtOH #2 few dips (10-15 sec)16. 95% EtOH +Glacial Acetic Acid 3 min17. 100% EtOH #1 1 min18. 100% EtOH #2 1 min19. Xylene #1 1 min20. Xylene #2 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

CoverslippingAfter completion of the staining process

1. Place a small drop of resin on a coverslip2. Drain off the xylene being careful not to let the slide dry3. 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 slidesSlides 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 regionsfor better comparison. In your report don’t forget to add labels, and label any ar tifacts. I am extending thedeadline for the lab reports to 4/4/14 so everyone will be able to take pictures