Cryostat protocol

The first method relies on cutting cryostat-, paraffin-, or lesin-embedded sections that are mounted alternately on two (or more) different microscope slides. Preferably, the sections should have a thickness of about 5 pm or thinner to increase incidence of portions of cell bodies in the adjacent sections. The approach with alternating sections can utilize any standard ICC protocol for each of the sets of sections One of the advantages with alternating sections is that each slide can be exposed to totally different ICC protocols optimized for the peptides to be detected. 

The procedures outlined in this section may be applied without modification when immimolabeling cryostat sections. Notes are cited to explain modifications of the protocols that may be applicable when immunolabeling vibratome sections, wholemounts of retina, wholemounts of invertebrate ganglia, or cultured cells. 

Full mechanistic details of asymmetric epoxidation (AE) reactions can be found in a comprehensive review. The features of the transition state which leads to high enantioselectivities over such a wide range of allyl functions have been intensively studied, but it is arguably more instructive from a practical point of view to indicate the behaviour of some commonly encoim-tered substrates with this catalyst. Tri- and tetra-substituted allylic alcohols with their electron-rich double bonds react rapidly, even at -35 °C. 3-( )-Monosubstituted allylic alcohols also react rapidly (1-4 h, as in Protocol 1) while other mono-substitution patterns dramatically slow down the reaction (10-50 h), necessitating the use of cryostatic cooling units. These reactivity patterns are summarised in Scheme 1.2. 

Embed up to approximately 12 heads in partially frozen O.C.T. Tissue Tek Compound on a cryostat chuck and orient accordingly using fine forceps as described in step 7 in Protocol 13.2. 

Coronal cryostat sections (10 pm thick) are cut after cryoprotec-tion and processed for indirect IMF. Sections are first incubated for 60 min with 10 % NGS or 10 % normal donkey serum in PBS to block nonspecific binding. They are then incubated with antibodies diluted according to manufacturers recommendations 1 h with primary antibody and then 1 h with secondary antibody diluted 1 100. Double IMF is performed according to standard protocols [48]. Mounting media contain DAPI to stain nuclei. Negative controls are performed each time immunostaining was done and consisted of preincubation with PBS, substitution of nonimmune isotype-matched control antibodies for the primary antibody, and/or omission of the primary antibody. Further details can be found in [48]. 

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