Scribd
Upload
26 views

Dissection Protocols

This document provides protocols for dissecting tissue, staining tissue samples, and mounting stained tissue samples onto microscope slides for viewing. The dissection protocol instructs how to use tweezers to tear small pieces of tissue and transfer them to a sieve using a pipette. The staining protocol describes incubating tissue sections in thioflavin S and Hoechst solutions to stain amyloid plaques and cell nuclei. The mounting protocol instructs how to transfer stained tissue from the sieve onto a slide, apply a coverslip using fluoromount, and let the sample dry before viewing under a microscope.

Uploaded by

Andrea
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
26 views

Dissection Protocols

This document provides protocols for dissecting tissue, staining tissue samples, and mounting stained tissue samples onto microscope slides for viewing. The dissection protocol instructs how to use tweezers to tear small pieces of tissue and transfer them to a sieve using a pipette. The staining protocol describes incubating tissue sections in thioflavin S and Hoechst solutions to stain amyloid plaques and cell nuclei. The mounting protocol instructs how to transfer stained tissue from the sieve onto a slide, apply a coverslip using fluoromount, and let the sample dry before viewing under a microscope.

Uploaded by

Andrea
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
You are on page 1/ 2

Dissection Protocols

Equipment: Tweezers (S) – 2, Tweezer (L), Culture plate, PBS x1, 12-well plate, sieves, marker, 1000
uL micropipette, bleach bottle.

Use the two (S) tweezers to tear small pieces from around the tissue
a. Hold the tongs so the pointed tips are facing towards you and are flat against the tissue.
Pull UNIFORMLY around the tissue.
b. Tissue pieces should be small and you will be trained in how exactly the size will be in
person. Around 20 small pieces is enough. Your tissue pieces should still be visible to the
eye.
c. CPECs tissue is relatively firm, don’t be rough but don’t be afraid to tear the tissue.
Take your 1000uL pipette and load with plastic tip. Cut the plastic tip end w/ razor blade.
Using your modified pipet tip, transfer the tissue to the sieve. Try not pipet too much at once as the
tissue easily gets stuck to the plastic
a. Do not be afraid to lots of PBS, just don’t overflow the sieve
b. If a tissue piece is stuck in the plastic tip, pipet in and out PBS in the culture plate till it is
dislodged. Afterwards, pipet the dislodged tissue again with only a small amount of
solution and transfer.
c. Repeat transfer until all sample(s) are transferred to the appropriately labeled sieves in
your well plate
Disinfect your tweezers with bleach and dry with kim wipes. Spray your culture plate with PBS with
bleach and leave for a few minutes. Do not dump into sink, just throw into biohazard waste. Also
throw any kimwipes to dry into biohazard along with your used pipet tip when finished.

Staining Protocols [Belly Dancer = 3-4, Wash = 3mL, Stain = 2mL]


1.
a. 1x PBS (5 min)
b. 50% EtOH x 2 (5 min)
2. Incubate sections in 0.5% Thioflavin S solution in 50% EtOH  (0.05g in 10 mL 50% EtOH).
Cover with foil. (10 min)
3. 50% EtOH  x 2 (10 min)
4. PBS 1x x 3 (10 min)
5. 2ug/mL Hoechst (10 min)
a. Stains nuclei of our tissue, similar to DapI stains.
6. PBS 1x x 2 (5 min)
7. If you don’t have time to mount and coverslip samples, PBS-A, parafilm, foil. Leave in fridge,
label date, initial.
Mounting Protocols

1. Use the 1000 uL pipet. AVOID making BUBBLES as they are hard to remove from a thick
solution. Press the pipet tip against the wall of the container when pouring back solution to reduce
bubbles. When extracting Fluoromount G, place pipet far below the surface of the solution and wait for the solution
to reach the pipet level as pipetting too fast introduces bubbles.
a. If you have too many bubbles, try using the vacuum to extract the bubble from beneath the coverslip. Use the
“Superfrost Plus” Microscope slides. The side you should be placing samples on is the “rough” side where
you can feel the surface of the white label tip.
2. Label each slide: Sample, HuCHP, ThioS + Hoechst, Date, Initials.
3. If use 2 spacers (be very careful, $1 a spacer): use forceps to peel, stick 2 layers down. DO NOT
unpeel the sticker on top unless coverslip is ready.
4. Take a new culture dish and fill with 2/3rds with PBS. Add a small amount of PBST.
5. Wet the surface with PBS/PBST solution from the dish using a 1.0 paintbrush.
6. Tilt the sieve to get the tissue pieces to one side of the sieve, use a paintbrush and sweep out the
tissue from the tissue onto the plate.
7. Spread out tissue into a grid-like fashion so each piece is separate from one another.
8. Take a 1000uL micropipette and extract 1mL Fluoromount G. Pour several drops onto the slide
and mount the coverslip or use 75-85 uL for 2 spacers.
a. When setting the coverslip, only touch the corners of the glass slip and slowly let down onto the slide. Try to
avoid bubbles as best you can. Do this by setting the slip slowly. It’s easiest to hold the cover slip corners or
the edges. If there are bubbles, try to ensure that they are not directly on the tissue as this reduces visibility
under the microscope. If there are bubbles, try to remove with a vacuum.
9. Let the sample dry IN THE DARK.

You might also like