Improvement in the evaluation of Fos-neuropeptide colocalization by employing free-floating cryosections of delicate thickness: double and triple colored immunohistochemistry

OBJECTIVE: The present study was aimed to select a methodical approach to optimize the thickness of cryo-processed free-floating sections for precise recognition between a single Fos signal and Fos/neuropeptide colocalizations in sequential double or triple colored immunohistochemical stainings. For this purpose brain sections of variable (5-20 microm) thickness were tested utilizing enzyme-substrate detection system employing oxytocin (OXY) and vasopressin (AVP) antisera.

METHODS: The animals were perfused by fixative 90 min after i.p. administration of 5 ml of hypertonic saline (1.5 M NaCl) which was used to stimulate the hypothalamic osmosensitive neurons. The brains were removed, soaked with 30% sucrose in 0.1 M PBS, cryo-sectioned throughout the hypothalamus into 5, 10, 15, and 20 microm thick coronal sections, collected and washed in 0.2 M glycine buffer for 10-15 min, and finely stored in 0.1 M PBS. Single Fos and Fos/OXY and Fos/ OXY/AVP colocalizations were processed employing avidin-biotin-peroxidase (ABC) complex and diaminobenzidine chromogen with or without adding Nickel chloride salt as a black and blue color inducer. Evaluation of the Fos-neuropeptide co-labeled perikarya manifestation was performed on a computerized Leica light microscopy.

RESULTS: The present data demonstrate that cryoprocessing enables generate free- floating sections of 5, 10, 15, and 20 microm thickness. Except the 5 microm thickness, all the other sections sizes tested exhibited well preserved tissue stability and excellent immunohistochemical properties either for single Fos reaction or double Fos/OXY and triple Fos/OXY/AVP costainings.

CONCLUSIONS: We adapted and optimized Fos immunohistochemistry for use of fixed and cryocut processed free-floating brain sections. The present data indicate that except 5 mum thickness all the other sorts of cryosections tested were sufficiently resilient for performing a sequential double or triple colored immunohistochemical stainings. However, 10 mum thickness reached the borderline of the handling safety, therefore, 15 mum section thickness will be the thickness of the choice recommended, which gave relevant immunoreaction, retained good tissue preservation, and ensured an appropriate clarity for accurate recognition between a single and colocalized Fos signals.