Fv10 asw software

Kidneys were snap frozen in OCT compound (Sakura Finetek, Torrance, CA) at the time of harvest and stored at −80°C. Four μm sagittal sections were cut using a cryostat, warmed to room temperature, and fixed with acetone. Non-specific binding was blocked with 10% heat-inactivated goat serum. Primary antibodies were diluted in 2% heat-inactivated goat serum and incubated overnight at 4°C. Autofluorescence was blocked with 0.05% Sudan Black B in 70% ethanol for 20 minutes at room temperature.^{53 (link)} High-resolution images were obtained with an Olympus FV1000 FCS/RICS confocal microscope (Olympus Scientific Solutions Americas Corp, Waltham, MA). The localization of deposits in the mesangium or in peripheral capillary loops was assessed based on the pattern of deposition. The intensity and co-localization of proteins were measured by drawing regions of interest (ROIs) around glomeruli and analyzing the ROIs with Olympus FV10-ASW software. Staining using isotype control antibodies is shown in Supplemental Figure S6.

Kidneys were snap frozen in OCT compound (Sakura Finetek USA Inc., Torrance, CA) at the time of harvest and stored at −80°C. Four μm sagittal sections were cut using a cryostat, warmed to room temperature, and fixed with acetone. Non-specific binding was blocked with 10% heat-inactivated goat serum. Primary antibodies were diluted in 2% heat-inactivated goat serum and incubated overnight at 4°C. Autofluorescence was blocked with 0.05% Sudan Black B in 70% ethanol for 20 minutes at room temperature, followed by two 10 minute washes in deionized water [35 (link)]. High-resolution images were obtained with an Olympus FV1000 FCS/RICS confocal microscope (Olympus Life Science, Waltham, MA). Olympus FV10-ASW software was used for intensity and co-localization analyses.

The mouse cell lines 4T1 isolated from mammary gland were used for the evaluation of polymer conjugate uptake using confocal laser scanning microscopy. Fluorescently labeled polymers, HD-P+Dy-633, LD-P-30+Dy-633 and LD-P-45+Dy-633, and polymer-drug conjugates, HD-P+Dox/Dy-633, LD-P-30+Dox/Dy-633 and LD-P-45+Dox/Dy-633, all with covalently bound Dyomics-633 were incubated with 4T1 cells for 24 h in a 5 % CO₂ atmosphere at 37 °C. The amount of the polymer conjugates added to the cell suspensions was normalized to the Dyomics-633 content (2 μg/mL). The normalization to the dye was used to evaluate the difference in internalization rate between the polymers and polymer conjugate. After incubation, the cells were washed two times with PBS and the cells were labeled with Hoechst 33342 in PBS (5 ug/mL). The polymer-bound dye Dyomics-633 was excited at 647 nm and the emitted light was detected through 650 – 750 nm filter. Hoechst 33342 dye labelling the nuclei was excited at 405 nm and emitted light was detected through 425 – 500 nm filter. The drug Dox was exited at 488 nm and emitted light was detected through 500–600 nm filter. The laser scanning confocal microscope Olympus IX83 with FV10-ASW software (Olympus, Czech Republic) was used to observe the fluorescence and transmitted light. The samples were scanned with a 60× oil immersion objective Plan ApoN (1.42 numerical aperture).