Additional file 10 of A pipeline for targeted metagenomics of environmental bacteria

Additional file 10: Figure S10. Signal intensity of formaldehyde fixed Gramella forsetii cells, after HCR-FISH with different treatments. CARD-FISH and the former protocol (yellow bar: in hybridization buffer A, no denaturation, 45 min amplification) were used for comparison. 30 min denaturation at 65°C and 85°C were tested in combination with hybridization buffer A and hybridization buffer B for three amplification times: 15 min, 45 min and 120 min. The signal intensities were measured via microscopy and are given in RU. The choice of hybridization buffer did not make a significant difference to signal intensity, but HCR-FISH signals were slightly higher when buffer B was used (see materials and methods for details in composition). Increasing chain reaction amplification time from 15 or 45 min to 120 min enhanced fluorescence from 0.3 (15 min) and 0.4 RU (45 min) to 1.1 RU (average values).