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. 2016 Sep 29;11(9):e0163856. doi: 10.1371/journal.pone.0163856

Fig 7. Effect of different buffers and coverslip-mounting agents on fluorescence stability of Vivid and original Qdots.

Fig 7

Single MHCII immunofluorescence staining was done on 8 serial sections with either Qdot 565 or Qdot 655Vivid conjugated secondary Ab. After staining, the sections were imaged in the wash buffer used (A). Two control sections were then mounted in cytoseal (Qdot 655Vivid) or Qmount (Qdot 565) and imaged regularly until the next day (B). The remaining slides were left in the indicated wash buffer and imaged regularly for 5 1/2 hours (330 minutes); they were then coverslipped and imaged after mounting (380 minutes), as well as the next day (1270 minutes) (C & D). A: After a single staining round the fluorescence intensity of Qdot 655Vivid and Qdot585 did not vary significantly between the different wash buffers. B: The fluorescence intensity of Qdot 655Vivid mounted in Cytoseal and of Qdot 565 mounted in Qmount after a staining protocol using Bond Wash remained stable in mountant for at least 1270 minutes (p = 0.599, ANOVA), although the fluorescence intensity of Qdot 655Vivid had still reduced after mounting, compared with unmounted slides (** p<0.001, t-test). C &D: Qdot 565 was more stable than Qdot 655Vivid in all buffers, but especially in BKRA (Tukey pairwise comparisons with 95% CI; * p<0.05). D: Qdot 655Vivid signal in Bond Wash and TBS quickly became undetectable due to quenching and photobleaching, but this was significantly improved in BKRA (Bond Wash and TBS grouping separately to BKRA, using Tukey pairwise comparisons with 95% CI; * p<0.05). E: Representative images of the quenching. All images were taken 1270 min after the staining. 1st column: slides were mounted straight after the staining; 2nd to 4th column: slides were mounted after 330 minutes in the indicated buffer. Fluorescence images are shown with the same scale: 0–0.04 for Qdot 565; 0–0.18 for Qdot 655. The system is able to quantify intensity of fluorescence ranging over at least 3 logs; however the requirement to show the fluorescence images with the same scale means for comparison means some images may appear black, even though the quantification was within the compatibility of the system.