Procedure

Procedure

Procedure

1. Dehydration

The fixed tissues are dehydrated in this step using a graded series of alcohol as given below

  1. 30% alcohol – ½ hour
  2. 50% alcohol – ½ hour
  3. 70% alcohol – 1 hour (45 min for shrimp
  4. 90% alcohol – ½ hour
  5. 95% alcohol – ½ hour
  6. Absolute alcohol I – ½ hour
  7. Absolute alcohol II – ½ hour

2. Clearing

  1. Xylene I – ½ hour
  2. Xylene II – ½ hour

3. Impregnation

  1. Xylene : wax (2:1) – 30 minutes
  2. Xylene : wax (1:2) – 60 minutes
  3. Molten wax – 2 hours

4. Blocking: Take suitable containers (boats) for blocking the tissues and embed the tissue in a block of wax. The orientation of the tissues has to the looked into at this stage, care is taken not to allow any air bubble in the block.

5. Trimming and sectioning: Cut the block to required cube shape and fish it on the chuck of microtome and cut it using the mitrotome. The size of the sections may be fix 8 to 10 u in size. The sections are then floated on the water bath maintained at 60oC and collected on clean slides and dried on a slide warmer.

6. Staining procedure

  • Keep the slides with sections in oven at 60oC for melting of excess wax.
  • Put in xylene I for 5 minutes to dewax.
  • Change to xylene II for 2 minutes for complete dewaxing.
  • Change to alcohol – I for 1-2 minutes.
  • Change to 90% alcohol for 1 minute.
  • Change to 70% alcohol for 1 minute.
  • Change to 50% alcohol for 1 minute.
  • Wash in running tap water for 2 minutes.
  • Transfer to Haemotoxyline for 5-10 minutes.
  • Wash in tap water. Put in Eosin for 2 minutes.
  • Change to 30% alcohol for 1 minute.
  • Change to 50% alcohol for 1 minute.
  • Change to 70% and 90% alcohol for 2 mins each.
  • Next change to absolute alcohol.
  • Change to xylene I for 3 minutes.
  • Change to xylene II until cover slipped. Do not allow the slides to dry.
  • Mount using DPx.

Duration of staining in haemotoxylin (nucleus stain and eosin (cytoplas stain) has to be standardised by observing in the microscope after clipping in both the stains.

Last modified: Tuesday, 10 April 2012, 6:22 AM