Timm Staining Protocol

From Nikki Sunnen, BCM 4/09

Tissue Preparation Solutions

1) Sulfide Perfusate
Sodium sulfide nonahydrate (Sigma #208043) 2.9g
Sodium phosphate (monobasic monohydrate) 3.0g
De-ionized water 250ml
**mix until the solution turns clear. If you continue to mix past this point, it may turn
yellow again, but should still be okay to use.

2) 10% Neutral Buffered Formalin

3) Post-Fixative
Gluteraldehyde (25%) 10ml
Dextrose 24g
De-ionized water 70ml

Timm Stain Solution

The Timm stain solution is comprised of 4 different solutions. The recipes for
these 4 comprising solutions are listed below, but the Timm stain solution is:
1) Gum Arabic 120ml
2) Citrate buffer 20ml
3) Hydroquinone 60ml
4) Silver nitrate 1ml

1) Gum Arabic
Gum arabic 500g
De-ionized water 1000ml
***This will take 3-4 days to dissolve! Prepare once, then aliquot into 50ml tubes and
freeze at -20°C.***

2) Citrate Buffer
Citric acid 5.1g
Sodium citrate 4.7g
De-ionized water to 20ml

3) Hydroquinone Developer
Hydroquinone 3.4g
De-ionized water to 60ml
**takes some time to dissolve, so use constant stirring

4) Silver Nitrate Solution

Silver nitrate 0.17g
De-ionized water 1ml
Acquiring and Preparing Tissue
1) Perfuse anaesthetized mouse with sulfide perfusate:
a) Perfuse mouse with 1x PBS until blood runs clear and liver turns pale. Using a
gravity set-up, this takes ~5-10min.
b) Switch to the sulfide perfusate, and perfuse for at least 10min (using the gravity
set-up. It’s slow, but I recommend it, especially for younger animals). Indicators of a
good perfusion include initial limb twitching, blue/gray extremities, and a black liver.
c) Remove the brain. It should have a blue/gray tinge of color as well.
2) Fresh brain should be treated for a total of 60min in this perfusate, so place in
perfusate after removal for an additional ~45-60min.
3) Rinse brain in de-ionized water and transfer to 10% neutral buffered formalin. Brain
can remain in formalin for 1-3 days, but no longer.
4) Post-fix in gluteraldehyde solution for 1.5hrs. Do not fix any longer as this will reduce
the silver stain.
5) Return to 10% neutral buffered formalin for 24hrs.

Paraffin Embedding
1) Transfer brain to 70% EtOH for a minimum of 4hrs. Brain can be left for longer (even
a few days).
2) Switch to 95% EtOH overnight.
3) Switch to 100% EtOH for 2hrs.
4) Switch to fresh 100% EtOH for another 2hrs.
5) Switch to CHCl3 overnight.
6) Place in wax for 4hrs at 60°C.

Sectioning
1) Prepare the waterbath for sectioning by sprinkling a small scoop of gelatin and
chromium potassium sulfate sparingly into the waterbath. Allow it to dissolve.
2) Cut sections with a vibratome 10-15 microns thick and place in waterbath. Mount on
“+” slides.
3) Dry slides in incubator/oven (60°C) overnight.

Staining
1) Dewax slides in 3 changes of xylene, 3min each.
2) Hydrate to de-ionized water through 4 changes of 100% EtOH, 95% EtOH. Wash
well in 2 changes of de-ionized water.
3) Stain in preheated (26°C) Timm stain solution for 45min in dark cupboard at room
temperature, then for 20min at 60°C.
4) Wash in de-ionized water.
5) Counterstain in cresyl violet working solution for 5min.
6) Dehydrate through 4 1min changes of 100% EtOH.
7) Clean with 3 1min changes of xylene.
8) Mount in Cytoseal.