Isolated Pancreatic Islets: Features of Technology of Fixation and Embedding of Islets and of Insulin Staining in B-Cells
Journal Title: Biomedical Journal of Scientific & Technical Research (BJSTR) - Year 2019, Vol 19, Issue 2
Abstract
For investigations of effects of various chemicals or drugs on pancreatic β-cells it is preferably to use the tissue culture method of isolated pancreatic islets (PI) which possess two important advantages in compared with model in vivo: a) It is possible to investigate direct effect on β-cells; b) Opportunity to investigate effect of precisely defined concentrations of studied substance directly on the PI. We propose improvements of fixation, paraffin embedding and staining of sections of isolated pancreatic islets, which can significantly to improve the results. 16 neonatal rats 4-5 days old and 6 adult rats were used. Method of isolation of PI [1] from pancreas tissue was used: a) Pancreas of rats were treated by 2% solution of collagenase in Hanks solution 3 times for 3 minutes at temperature of +370C and pH = 7.33-7.40; b) Washing in cold Hanks solution for 30 seconds 3 times; c) Separation in the density gradient of Dextran or manual selection of islets; d) Washing in Hanks solution for 1 min. 2 times; e) Visual manual selection of isolated PI; f) Centrifugation 400-500 rpm per min.; g) Cultivation in medium 199 +5% bovin serum + 5.5% glucose for 6 hours; h) Fixation in Bouin liquid (picric acid sol.30 ml +40% neutral formalin 10ml + acetic acid 2 ml) for 45-60 minutes (24h fixation of pancreas tissue); i) Wash in 700 alcohol 2 times; 10) fill in paraffin; Staining of Paraffin Sections by Histochemical Methods for Staining of Insulin and Zinc-Insulin Complex in β-Cells as: 1) Aldehyde-fuchsine method; 2) Fluorescent Diethylpseudoisocyanine method 3) Immunohistochemical methods; and 4) High specific for Zinc fluorescent method of staining by 8PTSQ (8-para (toluenesulhonylamino) quinolin [2-10]. Recommendations for Procedure of Embedding of PI in Paraffin: a) Liquid paraffin is poured into a plastic tube (diameter 1 cm, height -2 cm) in a water bath at 560C; b) To collect suspension of PI into a pipette (or 1-2 ml syringe); c) Lower the end of the needle of syringe into paraffin at the level of 0.3-0.4 cm from the bottom of the tube; Slowly perform 2 actions: i. Squeezing the suspension into a tube with paraffin; ii. Simultaneous slow raising of syringe up to the level of 0.5 cm from the top of the tube; d) Then remove the tube from the water bath. Thanks to this Technology: 1) Islets are evenly located over the entire height of the paraffin block; 2) It is possible maximally prevent formation of air bubbles in the paraffin near the islets
Authors and Affiliations
Meyramov GG, Shaybek AS, Kartbaeva GT, Meyramova Abdraimova AG, Zhumagalieva ZZ
Keywords
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- EP ID EP620607
- DOI 10.26717/BJSTR.2019.19.003260
- Views 156
- Downloads 0
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