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HOW TO STAIN PFA-FIXED BRAIN TISSUE USING GOLGI-COX METHOD

Golgi–Cox method requires impregnation of fresh unfixed brain blocks. However, if PFA-fixed or formaldehyde-fixed tissues have to be used, some special treatments for fixed tissues should be performed to get good results.

1. The animal must be completely perfused with buffer and fixative. Perfusion should be performed within five minutes after death of the animal. Once tissues are removed, transfer them into the fixative as soon as possible. Fixed and sucrose solution treated tissues can be stored at -70°C for a long time.

2. Before Golgi-Cox staining, the fixed tissue needs to be placed under running tap water for at least 24 hours.

3. Transfer tissue into distilled water and store at room temperature in the dark for another 24 hours.

4. Transfer tissue into the impregnation solution and follow Tissue Preparation protocol from step 4 like unfixed Golgi-Cox brain tissues (Hito Golgi-Cox OptimStain™ Kit User Manual Page 6 – Standard Protocol or Page 8 – Vibratome Protocol) After the treatment, staining quality with fixed tissue is satisfactory for demonstrating dendritic branching pattern and dendritic spines. Glia cells can also be stained. Compared to the fresh tissues, PFA-fixed tissues or formaldehyde-fixed tissues have stained glia cells which contribute to higher background. Though we do not recommend using fixed tissues, our HITO GOLGI-COX OPTIMSTAIN™ KIT provides an alternative approach to do research with fixed pathological samples.

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How to make gelatin coated slides for Golgi staining?

Gelatin coated slides can hold Golgi sections on slides and prevent sections falling off. Using Hito Gel-Coat™ Solution for Golgi Staining, everyone can make low-cost gelatin coated slides with high quality like commercial grade products.
Procedure: Heat the water bath or oven to 50-60 °C, pre-warm the HITO GEL-COAT™ SOLUTION FOR GOLGI STAINING and allow the solution to completely liquefy, using a 50ml Coplin jar, dip slides (pre-cleaned) in the warm solution three times, drain the slides onto paper tissue, and then carefully stand the slides on end to air dry or in incubator at 37°C overnight. Store slides in slides box at room temperature.

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RESEARCH TOPIC- GOLGI STAINING PARAFFIN SECTION – RESEARCHGATE

RESEARCH TOPIC- GOLGI STAINING PARAFFIN SECTION – RESEARCHGATE
Link for a Research Topic – ResearchGate, our HITO GOLGI-COX OPTIMSTAIN™ KIT will be a solution

Is it preferable to do golgicox staining for brain in paraffin embedded tissue section?

https://www.researchgate.net/post/Is_it_preferable_to_do_golgicox_staining_for_brain_in_paraffin_embedded_tissue_section

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HOW TO PREPARE GOLGI STAINING SAMPLES USING PARAFFIN EMBEDDING METHOD

HOW TO PREPARE GOLGI STAINING SAMPLES USING PARAFFIN EMBEDDING METHOD

JANUARY 2, 2017 11:53:00 AM EST

How to prepare golgi staining samples using paraffin embedding method

  1. Prepare Hito Golgi-Cox OptimStain™ Kit Impregnation Solution
  2. Prepare animal for infusion by administering a lethal dose of anesthesia. Monitor it until the point when the animal fails to respond to pinching of the foot.
  3. Do not perfuse with buffer or fixative. Remove the tissue (brain, spinal cord or heart) as soon as possible.  Handle with care and avoid damage of the tissue. Large specimens should be sliced with a sharp blade into blocks of approximately 10 mm thickness.
  4. Rinse tissue in double distilled water for 2-3 seconds to remove blood from the surface.
  5. Transfer tissue into the mixed impregnation solution that is at least five times the volume of the tissue and store at room temperature in the dark.
  6. Replace the impregnation solution on next day (after 12-24 hours), and store at room temperature (20 – 25°C) for two weeks in the dark. To avoid non-specific staining, do not extend the impregnation time.
  7. Transfer tissue into Solution-3 that is at least five times the volume of the tissue. Store at 4°C in the dark. Replace Solution-3 after 24 hours, and continue to store at 4°C in the dark for 2-3 days.
  8. After 2-3 days processing in Solution-3, transfer the tissue into a new vial and wash the tissue with double distilled water overnight at room temperature.
  9. After water rinse, dehydrate the tissue in a graded ethanol/water series at room temperature. Clear tissue in chloroform for 12-24 hours. Process tissues in paraffin for 2-4 hours, preferably in a vacuum oven. Embed tissues in paraffin blocks (recommend paraffin with a lower melting point ). Note: Xylene can also be used as the clearing agent. The tissues cleared by chloroform will be softer and thus easier to cut. Using xylene as the clearing agent sometimes can lead to cracks of tissue sections.
  10. Turn on the water bath and set the temperature at 45-50°C. Use fresh deionized water. Insert the paraffin block into the microtome chuck. Set the dial to cut 50-80 µm sections. Cut sections very slowly and pick them up with forceps or a fine paint brush and float them on the surface of the water bath. Float the sections onto the surface of HITO DUAL-SAFE™ GELATIN-COATED SLIDE.
  11. Drain slides upright and dry at 37°C for a minimum of 15 minutes.
  12. Place the slides with paraffin sections in a 60°C oven for 2 hours to bond the tissue to the glass. Slides can be stored in a slide box at room temperature for three months.
  13. Deparaffinize sections in xylene, 3 times, 5 minutes each.
  14. Rehydrate sections in 100%, 95%, 70% and 50% ethanol 2 times, 5 minutes each.
  15. Rinse slides in distilled water 3 times, 3 minutes each.
  16. Mix 2 ml Solution-4, 2 ml Solution-5 and 6 ml double distilled water in a 12 ml staining jar (provided in the kit), then place slides in the solution mixture. Tightly close the staining jar and wait for 10 minutes.
  17. Rinse slides in distilled water 2 times, 4 minutes each (distilled water should be renewed frequently).
  18. Counterstain sections with cresyl violet (optional step).
  19. Dehydrate slides which was previously rinsed with distilled water, in 50%, 75% and 95% ethanol, 5 minutes each.
  20. Dehydrate slides in 100% ethanol, 3 times, 5 minutes each.
  21. Clear in xylene, 2 times, 5 minutes each, and apply coverslip over sections using undiluted xylene based resinous mounting medium.
  22. Allow to dry. The slides can be viewed after drying by bright field microscopy.

paraffin section / golgi cox staining