We used to stain mossy fibers in hippocampal tissue. Unfortunately we faced unspecific staining all over hippocampal areas. Therefore we are looking for a way to remove or prevent this type of interference.
I am really no expert for Timm staining, but made a short look at the protocols on www. A simple way to avoid the background should be to make several sections, that are incubated for different times and then pick that one, with the best noise-to-signal ratio.
Silver stains are usually very hard to remove again from tissue. In clinical histology we never did it, as long as we had enough material left for new sections. For silver stains it is critical to observe the reaction in short intervals. Often there is a "point of no return". first the slide looks nearly unstained and a few minutes later it is black. : (
Hello,
you are sure that is not an artifice from the procedure previous to staining. For example, bad fixation, bad cleared, bad impregnation, etc...Because you say "...Unfortunately we faced unspecific staining all over hippocampal areas..."
If ultimately, was not artifice, maybe you need realizing decolorization (time) or absorption (time) of the silver nitrate.
If you consult somebody chemist may give you better help in reactions, and histochemical with or not hot.
Also, I am no expert toTimm staining, but also made a short look at the protocols on some reactions:, thus, only is a suggestions that you need prove...
and if you will use activated Charcoal ? and/or Copper(II) sulfate ?..
I found in internet:
A typical reaction with silver nitrate is to suspend a rod of copper in a solution of silver nitrate and leave it for a few hours. The silver nitrate reacts with copper to form hairlike crystals of silver metal and a blue solution of copper nitrate:
2 AgNO3 + Cu → Cu(NO3)2 + 2 Ag
Aqueous silver nitrate also decomposes when heated:
2 AgNO3(aq) → 2 Ag(s) + O2(g) + 2 NO2(g)
Most metal nitrates thermally decompose to the respective oxides, but silver oxide decomposes at a lower temperature than silver nitrate, so the decomposition of silver nitrate yields elemental silver instead.
best regards,
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.
- A novel non-perfusion Timm method for human brain tissue.
Dick Jaarsma, Jakob Korf
Correspondence: Dick Jaarsma, Department of Biological Psychiatry, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands.
Abstract
A simple modification of the Timm sulphide-silver method for unfixed brain tissue is described. Instead of perfusing animals with a sodium sulphide solution, sulphide treatment was performed by exposure to mounted frozen sections to H2S gas. The staining pattern in the rat brain obtained with this modification is similar to that With the Neo-Timm method, primarily yielding staining of the neuropil of telencephalic structures. Because perfusion is not required, the modified Timm method can also be applied to postmortem human tissue. Application of the modified Timm stain to the human tissue is exemplified on the hippocampus with special attention to the non-mossy fiber staining.
I am having problem with Gum Arabic in the staining protocol, after staining I observed the gum arabic is sticking to the tissue and also in between spaces. Is there a way to get rid of this ? I am using 50% Gum arabic
To be honest: cf. also RG-Thread:
https://www.researchgate.net/post/Can_someone_advise_on_Timm_staining_protocol
and - especially @Raghava Jagadeesh - find information: Fabio Nobili (his comment #02] describes in one of his recipes mentioned in that thread the use of only 20% gum arabic.
Additionally to the alternative method mentioned by Egidio Stigliano in this thread [comment # 06] regarding an alternative to the use of sodium sulphide solution by perfusion fixation, Christopher J. von Ruhland in the other thread ['Can_someone_advise_on_Timm_staining_protocol', comment #05] reports about / proposes to use a method not using gum arabic:
cit.:"....central to the method: use of a reducer and silver salt together in
solution (so-called physical development)." [end of citation from the article*)]
Geoffrey R. Newman, Bharat Jasani (1998) 'Silver Development in Microscopy and Bioanalysis: a New Versatile Formulation for Modern Needs'. The Histochemical Journal 30(9) 635-645; abstract (and first two pages of the article) available at the journal's website: http://link.springer.com/article/10.1023/A%3A1003404128497 ,
*) if you should be in need of that article urgently I could provide the article (which I cannot attach here for copyright reasons). For such an action please send your requesting message either via RG-messaging system or via [email protected] to me, mentioning at least: "Geoffrey R. Newman, Bharat Jasani (1998) Silver development"
Regards, W.M.
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