Evaluation of Long-Term Cryostorage of Brain Tissue Sections for Quantitative Histochemistry

Abstract

Storage of tissue sections for long periods allows multiple samples, acquired over months or years, to be processed together, in the same reagents, for quantitative histochemical studies. Protocols for freezer storage of free-floating frozen sections using sucrose with different additives have been reported and assert that storage has no effect on histochemistry, but no quantitative support has been provided. The present study analyzed the efficacy of long-term storage of brain tissue sections at -80C in buffered 15% glycerol. To determine whether histochemical reactivity is affected, we analyzed 11 datasets from 80 monkey brains that had sections stored for up to 10 years. For processing, sections from multiple cases were removed from storage, thawed, and batch-processed at the same time for different histochemical measures, including IHC for neuronal nuclear antigen, parvalbumin, orexin-A, doublecortin, bromodeoxyuridine, the pro-form of brain-derived neurotrophic factor, and damaged myelin basic protein as well as a histochemical assay for hyaluronic acid. Results were quantified using stereology, optical densitometry, fluorescence intensity, or percent area stained. Multiple regression analyses controlling for age and sex demonstrated the general stability of these antigens for up to a decade when stored in 15% glycerol at -80C.

A Survey of White Matter Neurons at the Gyral Crowns and Sulcal Depths in the Rhesus Monkey

“…Blocks were flash frozen at −75°C and stored at −80°C until sectioned at 30 μm thickness into 10 repeating series of sections. Series not processed immediately were stored in cryoprotectant (15% buffered glycerol) at −80°C until removed, thawed and batched processed for immunohistochemistry (Estrada et al, 2017 ).…”

Treatment with Mesenchymal-Derived Extracellular Vesicles Reduces Injury-Related Pathology in Pyramidal Neurons of Monkey Perilesional Ventral Premotor Cortex

“…At ;14-16 weeks after injury, monkeys were perfused using our twostage Krebs-PFA perfusion method for harvesting live tissue and subsequent fixation (Amatrudo et al, 2012;Estrada et al, 2017). The animals were initially sedated with ketamine hydrochloride (10 mg/ml) and deeply anesthetized with sodium pentobarbital (to effect, 15 mg/kg, i.v.…”

“…The brain was cut on a freezing microtome in the coronal plane at 30 or 60 mm. Sections were stored in cryoprotectant (15% glycerol, in 0.1 M PB, pH 7.4) at À80°C until use (Estrada et al, 2017).…”

Cell based therapy enhances activation of ventral premotor cortex to improve recovery following primary motor cortex injury

“…The 60 μm series was immediately mounted on microscope slides and stained with thionin for lesion reconstruction. The other series were collected in phosphate buffer with 15% glycerol and stored at −80°C for later histochemical processing (Estrada et al, 2017).…”

“…sections spaced 1200 μm apart) through the premotor cortices were removed from storage and thawed for c-Fos and synaptophysin immunohistochemistry. All the sections for each marker were batch-processed (see Estrada et al, 2017 for discussion of batch processing) in the same reagents at the same time, according to the following steps. First, sections were rinsed in 0.05M Tris-buffered saline (TBS) to remove the glycerol.…”