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Histology Fundamentals

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Suggested procedures for processing fixed tissue

Here are a few guidelines which you can use to process fixed tissue using Histo-Clear and Histo-Clear II:

Histo-Clear Automated Tissue Processing Schedule

Process Bath 18 Hour Cycle (Time in Hours) 24 Hour Cycle (Time in Hours)
70% ethanol 1.5 2
80% ethanol 1.5 2
95% ethanol 1.5 2
Absolute alcohol 1.5 2
Absolute alcohol 1.5 2
Alcohol/Histo-Clear (v/V) 1.5 2
HIsto-Clear 1.5 2
Histo-Clear 1.5 2
Histo-Clear 1.5 2
Paraffin 1.5 2
Paraffin 1.5 2
Paraffin 1.5 2

Histo-Clear II Tissue Processing Protocol
Process Bath Time (Hours)
70% ethanol 1.5
80% ethanol 1.5
95% ethanol 1.5
Absolute alcohol 1.5
Absolute alcohol 1.5
Anhydrous isopropanol (optional) 1.5 (may help to remove last trace of water)
10% ethanol, 90% Histo-Clear II 4
Histo-Clear II 4
Histo-Clear II 4
Paraffin 1.5
Paraffin 1.5
Paraffin 1.5
Paraffin 1.5

Histo-Clear II Dewaxing and Staining Protocol
Process Bath Time (minutes)
Histo-Clear II 5
Histo-Clear II 5
10% ethanol, 90% Histo-Clear II 5
Absolute alcohol 5
Absolute alcohol 5
95% ethanol10 5
80% ethanol 5
70% ethanol 5

 

For best results with Histo-Clear II:

  1. Rotate Histo-Clear II after every 3-5 runs for heavy use, or every 5-7 runs for light use.
  2. Replace the final absolute ethanol bath frequently, or at the first sign of water contamination. Histo-Clear II is very water intolerant. Including an isopropanol step before the first Histo-Clear II station and/or adding 10% ethanol to the first Histo-Clear II station will minimize the effects of water carryover (see protocols above).
  3. Mounting medium: Use a compatible medium, such as Omnimount (HS-110). Histo-Clear II is not compatible with DPX or Histomount.

Note: These protocols are recommended starting points for developing an optimal process for a given lab's equipment, tissue types, section thickness, etc.

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