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The page below is a sample from the LabCE course Troubleshooting Guidance for Hematoxylin and Eosin (H&E) Stain. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Nuclear bubbling can be caused by poorly buffered fixatives.

Sample Fixation

Samples should be collected and immediately immersed in fixative to ensure proper fixation. Formalin is the most commonly used fixative in the laboratory and does provide good nuclear detail. However, formalin should be treated as would any other reagent, specifically with regard to storage and expiration dates. Leaving formalin in direct sunlight can alter the pH of the solution, causing an acidic environment for tissues. Acidity in fixatives tends to have two primary impacts on tissues:
  1. Searing of the outer edges of the tissues
  2. Overdrying of the tissues
Searing occurs when the outer edge of the sample had been altered to give a "burned" appearance. This change also prevents proper infiltration of reagent into the sample because the proteins along the outer portion of the sample create a barrier for reagent to enter and for water to leave.
Depending on the level of acidity, the tissues can merely become overly dry. Whenever concerns about formalin viability arise, it is best to start with fresh reagent to reduce the risk of tissue damage.