Improved cryosections and specific immunohistochemical methods for detecting hypoxia in mouse and rat cochleae.
The present study was undertaken to develop an improved cryoembedding method for analysis of mice and rat cochleae, which permits high-quality cryosections and preserves overall structure and cellular resolution as shown by hematoxylin/eosin staining. The preservation of morphology and antigenicity is mandatory to achieve optimal results. A total of 20 male cd/1 mice and 14 male Sprague-Dawley rats were used in experiments for optimization of preservation, fixative, decalcification, embedding and cryosectioning of cochleae from adult and aged rodents. In addition, a novel immunohistochemical procedure (using Hydroxyprobe-1 kit) was developed for detecting regions of hypoxia in mice and rat cochlea. This method employs a primary fluorescent-conjugated monoclonal antibody directed against pimonidazole protein adducts that are created in hypoxic tissues. Subsequent studies of hypoxia inducible factor-1alpha (HIF-1alpha) by immunofluorescence in the cochlea of these animals were performed in order to confirm that immunochemical detection of pimonidazole protein is representative of a hypoxic environment. We conclude that the present method results in high-quality cryosections of cochlear tissues presenting good anatomical and histological preservation. Furthermore, our optimized procedures provide novel tools for the investigation of neuro-sensory-epithelium in physio-pathological situations associated with hypoxia and/or ischemia, such as inner ear development, plasticity, regeneration and senescence.
Donadieu
Emilie
E
Laboratoire d'Otologie Neuro-otologie et Micro-Endoscopie, IFR Jean Roche, Faculté de Médecine Nord, Université de la Méditerranée, Bd Pierre Dramard, 13916 Marseille cedex 20, France.
Hamdi
Wissal
W
Deveze
Arnaud
A
Lucciano
Michel
M
Lavieille
Jean-Pierre
JP
Magnan
Jacques
J
Riva
Catherine
C
D000818:Animals; D021461:Animals, Outbred Strains; D015415:Biomarkers / Q000378:metabolism; D015834:Cochlear Diseases / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D019084:Fluorescent Antibody Technique, Indirect; D005629:Frozen Sections; D000860:Hypoxia / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D008297:Male; D051379:Mice; D009593:Nitroimidazoles; D051381:Rats; D017207:Rats, Sprague-Dawley; D016610:Tissue Embedding / Q000379:methods
D015415:Biomarkers; C497443:Hif1a protein, mouse; C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009593:Nitroimidazoles; C033815:pimonidazole