pk 4000 hematoxylin qs counterstain vector laboratoires cat  (Vector Laboratories)

Journal: Science Advances

Article Title: A long-term ketogenic diet causes hyperlipidemia, liver dysfunction, and glucose intolerance from impaired insulin secretion in mice

doi: 10.1126/sciadv.adx2752

Figure Lengend Snippet: ( A to G ) Analysis of chromogenic insulin/proinsulin staining in the pancreas with hematoxylin counterstain. Qupath was used to quantify islet area based on detection of the brown insulin stain. [(A) to (C)] Males, 8 to 12 slices were taken throughout the pancreas from n = 4 to 6 animals per groups for a total 5157 islets analyzed on LFD, 6718 on HFD, and 6389 on KD. [(E) to (G)] Females, six to seven slices were taken throughout the pancreas ( n = 5 per group), for a total of 3749 LFMP and 3428 KD islets analyzed. [(A) and (E)] Average islet area. [(B) and (F)] Frequency distribution of islet sizes as number of islets per slice in each bin. Bins correspond to islet diameter 100 μm 2 = diameter of 11.3 μm or the size one β cell (and the minimum size needed to identify a nucleus and clear borders in the analysis); 170 μm 2 = diameter of 14.7 μm; 490.87 μm 2 = diameter of 25 μm; 1963 μm 2 = diameter of 50 μm; 4417.86 μm 2 = diameter of 75 μm; 7853.981 μm 2 = diameter of 100 μm; and 17,671 μm 2 = diameter of 150 μm. [(C) and (G)] Average number of islets per slice. (D) Representative images from male islets. Scale bar, 100 μm. ( H ) Male pancreas weight after 6 to 9 months on respective diets ( n = 17 to 18 per group pooled from three independent cohorts) and ( I ) insulin content after 6 months on the diets ( n = 5 to 6 per group). ( J ) Female pancreas weight ( n = 4 to 8 per group) and ( K ) insulin content after 6 months on respective diets ( n = 3 to 4 per group. Statistics: One-way or two-way ANOVA with Tukey’s HSD for post hoc testing was used. The number of symbols represents P values as: * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Data are represented as means ± SEM.

Article Snippet: After washing 3× for 5 min with PBS + 0.05% Tween 20 the horseradish peroxidase–conjugated insulin antibody was used (Abcam, catalog no. 28063 mouse anti-insulin and pro-insulin- AB dilution, 1:200) in the dark for 60 min. Antibody was washed off as described above and followed DAB application (Thermo Fisher Scientific, catalog no. 34002) and a hematoxylin counterstain (Vector Labs, catalog no. H-3502).

Techniques: Staining