Preservation of ultrastructure and immunoreactivity in cryosections of brain tissue stored in a sucrose-gelatin solution at freezing temperatures.
We evaluated the preservation of ultra-structure and immunoreactivity in cryosections of central nervous system tissue mounted with and stored in a sucrose-gelatin solution for one month at -20 degrees C or -80 degrees C. The ultra-structure of synaptic structure in these sections was well preserved and comparable to that of freshly cut cryosections. Quantitative analysis of mitochondrial ultra-structure demonstrated gradually lower degrees of preservation in sections stored at -20 degrees C and -80 degrees C compared with that in freshly cut sections. We observed distinct metabotropic glutamate receptor 1 (mGluR1)-immunogold labelling at peri-synaptic sites in freshly cut sections and also in those stored at -20 degrees C and -80 degrees C. Quantitative analysis of mGluR1 immunoreactivity revealed that the total number of immunogold particles per synapse and the number of non-specifically bound particles were similar under all three conditions. However, the percentage of gold particles bound to a specific synaptic region was greatest in freshly cut sections (79.0%) and progressively lower in sections stored at -20 degrees C (76.1%), in which sections were not frozen, and in sections stored at -80 degrees C (68.0%). These data indicate that ultra-thin cryosections may be conveniently stored in a sucrose-gelatin solution at -20 degrees C for cryoultramicrotomy-immunolabelling.
Akagi
T
T
Iwate Medical University-The Center for Electron Microscopy and Bio-Imaging Research, Lab for Nano-Neuroanatomy, Japan. akagit@iwate-med.ac.jp
Ishida
K
K
Tohno
H
H
Hanasaka
T
T
D000818:Animals; D002525:Cerebellar Cortex / Q000276:immunology / Q000648:ultrastructure*; D015925:Cryopreservation / Q000379:methods*; D016616:Cryoultramicrotomy / Q000379:methods*; D005615:Freezing; D005780:Gelatin; D007150:Immunohistochemistry; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D017470:Receptors, Glutamate / Q000378:metabolism; D012996:Solutions; D013395:Sucrose
D017470:Receptors, Glutamate; D012996:Solutions; D013395:Sucrose; D005780:Gelatin