In favor for the first explanation accounts that in the clinical setting the difference was consistent over a large number of biopsies.
Captisol ic50 The evaluation of the optimal number of reference genes needed to obtain reliable data strengthened the observation that the combination of a Menghini NaCl biopsy followed by RNAlater preservation and an RNAeasy mini kit extraction yields optimal RNA quality from canine liver biopsies. The size of the biopsy needle used in this study was based on a previous study on rat liver biopsy techniques, and turned out to be an optimal balance between quantity and quality of the biopsy and the health risks for the animal . This approach of RNA retrieval proved to be a rapid and feasible method for storage for further molecular analysis, and is in agreement with the findings of others for yeast, human renal and uterine myometrial tissues [15–17]. The quality of the obtained RNA in our approach was feasible Selleckchem TPCA-1 for micro-array analysis, which requires the highest possible RNA quality, preferential a RIN value above 8.0. Unfortunately our results show that optimal RNA stabilization was only achieved with media that were unsuitable for histology or immunohistochemistry. Histology of RNA later treated biopsies, evaluated in HE and reticulin staining turned out to be of insufficient
quality; furthermore, for the antibodies tested either the background staining was too high or central staining appeared very poor. The best fixative for (immuno)histochemistry proved to be 10% neutral buffered formalin. Boonfix fixation gave good morphology and results in routine HE and reticulin staining, but was suboptimal for the tested immunohistochemical staining methods.
RNAlater fixation yielded poor morphology in routine histology and in immunohistochemistry. Most likely, these shortages in morphological evaluation of RNAlater treated specimens were related to insufficient tissue fixation. Boonfix treated specimens generally evoked less intense reactivity immunohistochemically, but as all tested methods were optimized for use in formalin fixed (24 hrs) wedge biopsy specimens, Interleukin-3 receptor they might perform better in a study where the protocols are tailor-made to the fixative. Storage in minus 20°C for Boonfix and RNAlater, as required for molecular purposes, significantly worsened tissue morphology. In our experience staining artefacts more frequently occur in small formalin fixed paraffin embedded biopsies. We hypothesized that in the relatively small biopsies overfixation could easily occur. Therefore an RO4929097 chemical structure effect of the duration of formalin fixation was assessed with subsequent immunohistochemical evaluation of antibodies to proteins at three different (sub)-cellular locations in addition to routine histological staining methods. Differences of the immunohistochemical reactivity for all three antibodies were found between wedge biopsies and the smaller Menghini tissue samples in this study.