Cell experiment:

Cochlear sections containing SGNs are placed in 24 wells plates containing PBS (pH 7.4) and stored at 4℃. The sections are then used for Cresyl violet acetate and immunohistochemical (IHC) staining. Every 7th section is stained with Cresyl violet acetate (1%), dehydrated with ascending grades of alcohol, cleared with xylene, mounted with DPX and observed under microscope. Approximately 12-13 Cresyl violet acetate staining sections from each specimen are used for stereology. None of these cases show any histopathological changes under the light microscope. Estimation of the total volume of the Rosenthal canal (RC), total number of SGNs (optical fractionator probe) and the volume of the soma and their nucleus (nucleator probe) is done with software[1].

Cresyl violet acetate is a red fluorescent stain, which can be used to stain neurons.

The estimated total number of SG neurons is 27,485±3251 and 26,705±1823 in the PV and Cresyl violet acetate stained sections, respectively. There is no significant difference between them (p=0.552). Therefore, Cresyl violet acetate staining is simpler and more cost effective when estimates neuronal number. Although PV stains spiral ganglion neurons (SGNs) with a greater intensity and provides a functional status, its tedious protocol limits its use for quantification. Total RC volume is estimated using probe and it is found that an average RC volume of 2.162±0.35 mm3 and 1.82±0.33 mm3 in Cresyl violet acetate staining and PV immunostaining sections, respectively. Volume of neurons is estimated using nucleator probe and it is 3487.63±951 μm3 and 3740.1±784 μm3 in Cresyl violet acetate staining and PV immunostaining sections, respectively. Similarly, volume of neuronal nucleus is also estimated using nucleator probe and it is found to be 131.68±50 μm3 and 126.51±33 μm3 in Cresyl violet acetate staining and PV immunostaining sections, respectively[1].

[1]. Kaur C, et al. Comparison of unbiased stereological estimation of total number of cresyl violet stained neurons and parvalbumin positive neurons in the adult human spiral ganglion. J Chem Neuroanat. 2017 Jun 23. pii: S0891-0618(17)30037-6.