Peroxidase suppressor

Example 8

Tumour microarrays were made from biopsies obtained from 10 breast and 7 colon cancer patients. For each patient biopsy, four cores from different regions were arranged in an array. H&E staining of the TMA section confirmed that cytomorphological traits were representative of malignant tissue, as opposed to stromal tissue. Each breast TMA contained 40 (4×10) tumour cores, representing 4 distinct areas of the same tumour biopsy for each patient. Each colon TMA contained 28 (4×7) tumour cores, representing 4 distinct areas of the same tumour biopsy for each patient.

Two identical TMAs were de-waxed and rehydrated, subjected to heat antigen retrieval by microwaving in TRIS-EDTA (pH 9.0) buffer, for 10 min at 800 W power.

TMAs were further incubated with fresh sodium borohydrate buffer for 10 min, followed by blocking with 1% BSA/PBS. TMAs were the incubated with peroxidase suppressor (Thermo Scientific Pierce) for 15 min. For the two-site TSA-FRET assay was performed as indicated above. Lifetime measurements were performed using both donor-labeled and donor plus acceptor-labeled TMAs, for each core. FRET efficiency calculations with a low signal-to-noise (signal lower than 4 times the background intensity) were excluded. The maximum FRET efficiency of four-regions of interest within the core was calculated as described above (FIGS. 5 and 6).

Example 8

Tumour microarrays were made from biopsies obtained from 10 breast and 7 colon cancer patients. For each patient biopsy, four cores from different regions were arranged in an array. H&E staining of the TMA section confirmed that cytomorphological traits were representative of malignant tissue, as opposed to stromal tissue. Each breast TMA contained 40 (4×10) tumour cores, representing 4 distinct areas of the same tumour biopsy for each patient. Each colon TMA contained 28 (4×7) tumour cores, representing 4 distinct areas of the same tumour biopsy for each patient.

Two identical TMAs were de-waxed and rehydrated, subjected to heat antigen retrieval by microwaving in TRIS-EDTA (pH 9.0) buffer, for 10 min at 800 W power.

TMAs were further incubated with fresh sodium borohydrate buffer for 10 min, followed by blocking with 1% BSA/PBS. TMAs were the incubated with peroxidase suppressor (Thermo Scientific Pierce) for 15 min. For the two-site TSA-FRET assay was performed as indicated above. Lifetime measurements were performed using both donor-labeled and donor plus acceptor-labeled TMAs, for each core. FRET efficiency calculations with a low signal-to-noise (signal lower than 4 times the background intensity) were excluded. The maximum FRET efficiency of four-regions of interest within the core was calculated as described above (FIGS. 5(a)-6(c)).

Example 6

For the acquisition of lifetime maps the mFD-FRET microscope was used as described above. SKBR3 cells were cultured in two 8-well chamber slides+/−stimulation or inhibition with EGFR and LY294002 respectively as described previously.

Following treatment, cells were fixed in PFA, permeabilized and blocked (1% BSA in PBS) for 1 h at room temperature. Cells were further incubated with peroxidase suppressor (Thermo Scientific Pierce) for 15 min to inhibit any endogenous peroxidase activity from cells.

The two-site FRET assay was performed as above. The main difference here was that cells on the second chamber slide were labeled using Alexa-594 (acceptor) TSA system for 15 min. For each experiment control cells labeled with only secondary Fab fragment conjugates were included. The labeled SKBR3 cells were mounted with ProLong Gold anti-fade reagent. A reference solution of Rhodamine B (in H₂O) was prepared and imaged prior to mFD-FLIM acquisition of the SKBR3 cells. The lifetime imaging experiments were performed as above. Three replicates were performed for each data point (FIG. 3(a)).