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