Crude trypsin

Manufactured by Thermo Fisher Scientific

Sourced in United States

Crude trypsin is a digestive enzyme derived from bovine pancreas. It functions to catalyze the hydrolysis of peptide bonds, specifically those involving the carboxyl group of lysine or arginine residues.

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Lab products found in correlation

In situ cell death kit, by Roche (2 mentions) Axiophot photomicroscope, by Zeiss (1 mentions) Apotome microscope, by Zeiss (1 mentions)

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Trypsin (6705 citations)

7 protocols using crude trypsin

Quantifying Diabetic Retinal Capillary Degeneration

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The retina from formalin-fixed eyes of ZDF and lean rats was rinsed overnight with running water, digested with 3% crude trypsin (Invitrogen-Gibco, Grand Island, NY, USA) containing 200 mM sodium fluoride. Trypsin-digested microvasculature was mounted on a glass slide, and stained with periodic acid Schiff-hematoxylin to count the degenerative capillaries.^{18 (link)}

Mishra M., Lillvis J., Seyoum B, & Kowluru R.A. (2016). Peripheral Blood Mitochondrial DNA Damage as a Potential Noninvasive Biomarker of Diabetic Retinopathy. Investigative Ophthalmology & Visual Science, 57(10), 4035-4044.

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Quantification of Retinal Microvascular Apoptosis

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Retina was isolated from the formalin-fixed eye, and rinsed overnight with the running water. The microvasculature was isolated by incubating the retina with 3% crude trypsin (Invitrogen-Gibco, Grand Island, NY) containing 200 mM sodium fluoride for 45 to 70 minutes at 37°C, and the neuro-retinal tissue was gently brushed away. The apoptotic vascular cells were detected by incubating the preparation with terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling stain (TUNEL; In Situ Cell Death kit; Roche Molecular Biochemicals, Indianapolis, IN). In each experiment, a positive control was run by exposing the retinal vessels to DNAse before initiation of the TUNEL reaction [9 (link),10 (link)]. TUNEL positive cells were identified in a masked fashion, and each trypsin digest was surveyed systematically under a Zeiss Axiophot photomicroscope.
After TUNEL staining, the microvasculature was stained with periodic acid-Schiff and hematoxylin for histologic evaluation. The number of acellular capillaries was counted in multiple mid-retinal fields (one field adjacent to each of the 5–7 retinal arterioles radiating out from the optic disc) and expressed as total acellular capillaries per retina [9 (link),10 (link)].

Kowluru R.A., Zhong Q., Santos J.M., Thandampallayam M., Putt D, & Gierhart D.L. (2014). Beneficial effects of the nutritional supplements on the development of diabetic retinopathy. Nutrition & Metabolism, 11, 8.

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Retinal Microvascular Degeneration Analysis

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The retina from formalin-fixed eyes was rinsed overnight with running water, and was incubated at 37°C with 3% crude trypsin (Invitrogen-Gibco, Grand Island, NY, USA) containing 200 mM sodium fluoride for 45 to 70 minutes. After gently brushing away the neuro-retinal tissue, trypsin-digested microvasculature mounted on a glass slide was stained with terminal deoxyribonucleotide transferase-mediated dUTP nick-end labelling (TUNEL) stain (In Situ Cell Death kit; Roche Molecular Biochemicals, Indianapolis, IN, USA). The microvessels were then rinsed, and stained with periodic acid Schiff-hematoxylin to count the degenerative capillaries [21 (link), 24 (link)].

Kowluru R.A., Mishra M., Kowluru A, & Kumar B. (2016). Hyperlipidemia and the development of diabetic retinopathy: Comparison between type 1 and type 2 animal models. Metabolism: clinical and experimental, 65(10), 1570-1581.

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Retinal Vascular Trypsin Assay

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The eyes were enucleated and fixed with 4% paraformaldehyde, and the whole retina was isolated and incubated in 3% crude trypsin (Gibco, Grand Island, NY) containing 200mM sodium fluoride at 37°C for 45–70 minutes. The neuroretinal tissue was gently brushed away under a microscope, and the vasculature was stained with terminal deoxyribonucleotide TUNEL using an In Situ Cell Death Kit (Cat. No. 11684795910, Roche Molecular Biochemicals). As a positive control, retinal vasculature treated with DNAse was also stained with TUNEL. The TUNEL-positive capillary cells were counted under a fluorescence microscope, and the microvasculature was then stained with periodic acid Schiff-hematoxylin to count acellular capillaries by light microscopy [22 (link), 26 (link)].

Mohammad G., Duraisamy A.J., Kowluru A, & Kowluru R.A. (2019). Functional Regulation of an Oxidative Stress Mediator, Rac1, in Diabetic Retinopathy. Molecular neurobiology, 56(12), 8643-8655.

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Retinal Vasculature Isolation and Quantification

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Whole retina was carefully isolated from the paraformaldehyde fixed eye, and incubated at 37°C for 45 to 75 minutes in 3% crude trypsin (Gibco, Grand Island, NY, USA) solution containing 200 mM sodium fluoride. The neuroretinal tissue was gently brushed away under a microscope, and the vasculature was stained with periodic acid Schiff–hematoxylin to count the acellular capillaries by light microscopy, and imaged using the Zeiss ApoTome microscope using a 40× objective.³ (link)^,²⁰ (link)

Mohammad G., Radhakrishnan R, & Kowluru R.A. (2020). Hydrogen Sulfide: A Potential Therapeutic Target in the Development of Diabetic Retinopathy. Investigative Ophthalmology & Visual Science, 61(14), 35.

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Retinal Vascular Trypsin Histology

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Retina isolated from the formalin-fixed eyes was rinsed overnight in running water and incubated at 37 °C with 3% crude trypsin (Invitrogen-Gibco, Grand Island, NY, USA) containing 200 mM sodium fluoride for 45 to 70 min. After cleaning the retinal vasculature under a dissecting microscope, it was stained with periodic acid-Schiff-hematoxylin to count acellular capillaries [24 (link), 31 (link)].

Malaviya P, & Kowluru R.A. (2024). Homocysteine and mitochondrial quality control in diabetic retinopathy. Eye and Vision, 11, 5.

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Retinal Capillary Degeneration Assessment

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Retina was isolated from the formalin-fixed eyes, and after rinsing overnight in running water, the retina was incubated for one hour at 37 °C in 3% crude trypsin (Invitrogen-Gibco, Grand Island, NY) supplemented with 200 mM sodium fluoride. The vasculature was cleaned under a microscope, and stained with periodic acid-Schiff-hematoxylin (PAS, Cat No. 395B, Sigma). Degenerative capillaries and pericyte ghosts were counted under a microscope^{27 (link),43 (link)}.

Kowluru R.A, & Mohammad G. (2020). Epigenetics and Mitochondrial Stability in the Metabolic Memory Phenomenon Associated with Continued Progression of Diabetic Retinopathy. Scientific Reports, 10, 6655.

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