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Techniques of celloidin removal from temporal bone sections.

Jennifer T O'Malley¹, Barbara J Burgess, Diane D Jones

¹Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114, USA.

The Annals of Otology, Rhinology, and Laryngology

|August 12, 2009

Summary

The best method for removing celloidin from mouse cochleae for immunostaining is using methanol saturated with sodium hydroxide. This technique ensures complete celloidin removal and yields clean, reproducible results for various antibodies.

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Area of Science:

Histology
Immunohistochemistry
Otolaryngology

Background:

Celloidin embedding is a common technique for preserving delicate tissues like cochleae.
Effective celloidin removal is crucial for successful immunostaining.
Previous methods for celloidin removal have shown variable efficacy.

Purpose of the Study:

To evaluate the impact of different celloidin removal techniques on immunostaining outcomes in embedded cochleae.
To identify the optimal protocol for celloidin removal to enhance immunostaining quality.

Main Methods:

Four celloidin removal protocols were compared: clove oil, acetone, ether-alcohol, and methanol saturated with sodium hydroxide.
Standardized tissue fixation, decalcification, and antibody application were maintained across all methods.

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Six antibodies targeting specific proteins (prostaglandin D synthase, Na+,K(+)-ATPase, aquaporin 1, connective tissue growth factor, tubulin, and 200 kd neurofilament) were used.

Main Results:

Clove oil, acetone, and ether-alcohol methods resulted in incomplete celloidin removal, compromising immunostaining.
The methanol-sodium hydroxide method effectively removed celloidin, leading to superior immunostaining.
This optimal method produced the cleanest and most reproducible staining for all tested antibodies.

Conclusions:

Methanol saturated with sodium hydroxide, diluted 1:2 with methanol, is the most effective solvent for celloidin removal in mouse temporal bone sections.
This optimized protocol ensures consistent and reproducible immunostaining results, critical for accurate histological analysis.