[Histonet] Apoptosis staining on mouse testis

Kant, H.J.G. van de (Henk) H.J.G.vandeKant <@t> uu.nl
Thu Aug 18 08:04:28 CDT 2011

Here a protocol for Bouin fixed tissue and it is working on testis.

TUNEL analysis on Bouin fixed, paraffin embedded tissue

  *   Incubation box in stove (37°C) with wet tissues.
  *   Dewax. All steps 3 min 3x Xylene, 2x Ehanol 100%, 1x Ethanol 96%, 1x Ethanol 70% and 1x MilliQ water.
  *   Microwave (700 Watt and at 98°C) 5 min in 10 mM Citrate buffer (pH 6.0).
  *   Put the slides directly in PBS on the shaker for 5 min.
  *   Incubate sections in 3% H202 in MilliQ water 5 min.
  *   Wash in PBS 3x5 min.
  *   Apply reaction mix:

Final concentration

1 section (30 ml)

1 section (40ml)

Amersham  buffer 1x


8 ml

Transferase 0.3U/ml

9U (0.75 ml)


dUTP 6.66 mM

0.2 ml

0.27 ml

MilliQ water (sterile)

23.05 ml

30.73 ml

For small sections apply 30 ml per section, 40 ml for bigger sections
NB! For negative control apply reaction mix without transferase

  *   Incubate 1h at 37°C in damp atmosphere

  *   End of reaction: Apply to each section the following buffer:
300mM NaCl, 30mM NaCitrate in MilliQ water and incubate for 15 min at RT

  *   Wash in PBS 3x5 min
  *   Blockage of non-specific sites with 2% BSA in PBS for 10 min at RT
  *   Apply ExtrAvidine 1:20 in 1%BSA PBS (30 or 40 ml/section) to sections and incubate 30 min at 37°C in damp atmosphere

  *   Wash in PBS 3x5 min

  *   Incubate in DAB solution (125 mgr DAB / 250 ml PBS + 85ml H2O2  37%) Incubate for 1 and 2 min. Use gloves and work in a fume cupboard.
  *   Wash in MilliQ water 5 min.
  *   Counterstain with Mayer for 30 sec.
  *   Dehydrate and mount with Pertex .

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