Coloração de tionina 0,2%

Nota prévia

Para esta coloração, após montagem das lâminas estas devem ficar 48h a secar a RT num ambiente “dust free”

1. Mergulhar em acetona ácida (5’)2. Escorrer e passar rapidamente por h2o destilada3. Corar na tionina TA (+/- 7’)4. Mergulhar em h2o dest5. Mergulhar em álcool ácido para diferenciar6. Passar em h2o destilada, secar com papel e colocar na estufa a 37ºC ate estarem bem

secas, depois deixar um pouco a TA7. Passar por xilol 85’) e montar com eukitt

Acetona acida 4partes de acetona para uma de ácido acético glacial

Tionina a 0,2%/formaldeído a 37%

Juntar 3 gotas de ácido acético glacial em cada 100ml, a solução deve ser filtrada antes de cada uso

A solução pode ser guardada depois de usada num frasco escuro em local escuro, durante bastante tempo

Álcool ácido

2partes de álcool isopropílico a 99% para uma parte de ácido acético glacial 10%

Thionin Stain, Modified Wisconsin

This stain is specific for DNA and Nissl substance, which is primarily ribosomal RNA. Thionin binds to acidic proteins and nucleic acids with a specificity determined by the pH of the final staining solution.

Working solutions of thionin should be filtered periodically and replenished when prolonged staining times (over 30 minutes) fail to produce acceptable stain intensity. Replenish by adding freshly filtered thionin working solution to replace about 10% of the solution in the staining dish (for example in a dish of 400 ml, replace about 40 ml).

I. Solutions.

A. Stock 1.3% thionin:

13 gm Thionin

1000 ml distilled H2O

Stir and heat gently for 1 hour. Filter the solution after the dye has dissolved. Store in a stoppered bottle. Use high purity thionin, such as Sigma T3387.

B. Buffers:

1. 1 M Acetic Acid (HAc):

58.5 ml glacial acetic acid

dilute to 1 liter with distilled water

2. 1 M Sodium Hydroxide (NaOH):

50 gm sodium hydroxide pellets

dissolve to 1 liter with distilled water

Mix buffer reagents together and adjust pH before mixing with the thionin stock in the formulas below.

II. Working Stains.

A. Full strength thionin stain (1%), 400 ml, pH 4.0.

80.0 ml 1M HAc

14.4 ml 1M NaOH

305.6 ml Thionin stock

This is the stain for routine Nissl stains. Staining time is usually from 30 seconds to 20 minutes.

B. Weak thionin stain (0.2%), 400 ml, pH 4.0.

80.0 ml 1 M HAc

14.4 ml 1 M NaOH

76.4 ml Thionin stock

dilute to 400 ml with distilled water

The weak solution is recommended for autoradiography slides, where the emulsion tends to encourage over-staining. Staining times vary from 30 seconds to 30 minutes with this stain.

Slides must be rinsed or rehydrated in distilled water before placing into thionin. Phosphate buffers, including PBS, will cause thionin to precipitate. This results in precipitate particles on your sections and a poorly soluble sludge in the bottom of the staining dish.

Always stain one slide as a test before committing an entire rack of slides to thionin stain. Inspect this test slide to determine if time in the stain solution should be changed. The staining action is influenced by choice of fixative, fixation time, embedment, section thickness, or section treatments.

The initial coloration will be purple, but the alcohols will "blue" the stained sections.

Remember to change your destaining solutions when you are through, be considerate of others. Dye-laden water and alcohol will interfere with the destaining. Residual thionin in the clearing alcohols may impart a background coloration that interferes with other people's slides.

Overstaining may be overcome by returning slides to a dish filled with 95% ethanol containing a few drops of glacial acetic acid. Dip slides into the acetic acid-alcohol until the color disappears - usually a matter of a few seconds. Then rinse them very well to remove the acid before restaining for a shorter time, or in the dilute thionin. Prolonged exposure to acetic acid-alcohol will prevent restaining. Contaminating the thionin with acetic acid-alcohol from destained slides will alter stain specificity.

The Purkinje cells of the cerebellum tend to exhibit the same thionin staining characteristics as the neurons of N. Magnocellularis. They may be used as an easily observable indicator of stain intensity when NM is not present in a test section.