Trypsin digestion method for the long-term formalin-fixed retinal in rats / 中国药理学通报

ABSTRACT

Aim To investigate the optimum trypsin digestion time and the optimum trypsin concentration of the long-term formalin-fixed retina in rats.Methods Male SD rats were sacrificed by cervical dislocation. The removed eyes were fixed in 4% formaldehyde fixa-tive for 2 days,2 weeks,4 weeks,3 months,5 months and 7 months respectively.Fixed retinas at dif-ferent time were separated and put in 3%,6% and 9% trypsin at 37℃ in incubator.The time required was recorded for complete digestion of retinas,which fixed for different time and digested in different con-centrations of trypsin.The retinal blood vessels were recorded under a microscope after PAS staining of stretched retinal blood vessel.Results After conven-tionally fixed for 48 hours,retinas were digested com-pletely in 3% trypsin at 37℃ for 3.5 hours.The di-gestion time extended to 9 hours and 1 4 hours for the 2 weeks and 1 month fixed eyes,using 3% trypsin, which achieved the same result as conventional fixed eyes.For the 3 months fixed eyes,a complete retinal blood vessel could also be attained by using conven-tional concentrations of trypsin,and only by extending digestion time to 22 hours.It is also indicated that the increased concentration of trypsin did not shorten the time of digestion.In addition,for the fixed eye from 5 months to 7 months,6% and 9% trypsin could short-en,to some extent,the time to 22 hours and 28 hours respectively,which was conductive to obtaining rela-tively complete retinal blood vessels.Conclusions Retinal tissues which fixed time shorter than 3 months can completely be digested using 3% trypsin.Howev-er,for retinal tissue,which fixed from 5 months to 7 months,both the extending time of digestion and the increasing concentration of trypsin to 6% are necessary to get optimum retinal vascular digestion.