Tcs sp2 laser scanning spectral confocal microscope

Double immunofluorescence staining was performed as described previously to explore the interaction between TLR₄ and TICAM-2 after IR [6 (link),7 (link)]. Briefly, 10-μm-thick sections were incubated with a primary rabbit anti-TLR₄ antibody (1:800; Abcam, Cambridge, United States) and a goat anti-TICAM-2 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, California, United States) overnight at 4°C. After incubation with an Alexa 488-conjugated donkey anti-rabbit immunoglobulin G (IgG) antibody (1:500; Molecular Probes, Eugene, United States) and an Alexa 594-conjugated donkey anti-goat IgG antibody (1:500; Molecular Probes), each for two hours at room temperature, images were captured using a Leica TCS SP2 laser scanning spectral confocal microscope (Leica Microsystems, Buffalo Grove, Illinois, United States).

On the day of the experiment, the volunteers were not allowed to eat or drink during the course of the tests. PL-biofilm samples collection was done individually (samples were taken from just one volunteer per day), starting at 8.30 AM (first baseline sample and immersions) and finished at 9.30 AM (mouthwash).
Immediately after the glass disks were withdrawn from the splints, they were submerged in 100 μL of fluorescence solution LIVE/DEAD® BacLight™ and kept in a dark chamber at room temperature for 15 min. A single investigator, masked to the study design, performed the microscopic observation using a Leica TCS SP2 laser scanning spectral confocal microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) with an HCX APOL 63x/0.9 water-immersion lens.

Sample collection was done individually at the Unit of Confocal Microscopy of the University of Santiago de Compostela at 8 am in the morning, so that the samples of each volunteer were analysed on different days. It was determined that a minimum of 10 hours should have elapsed since the last mouthwash on the previous night.
As the glass disks (in total, 6) were removed from the splint, they were immediately immersed in 100 μL of fluorescence solution LIVE/DEAD BacLigh and kept in a dark chamber at room temperature for 15 minutes. The characteristics of LIVE/DEAD BacLight fluorescence solution (Molecular Probes, Leiden, the Netherlands), as well as its preparation, have been described by the authors in a previous paper [36 (link)]. Microscopic observation was performed by a single investigator who was unaware of the study design, using a Leica TCS SP2 laser scanning spectral confocal microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) with an HCX APOL 63x/0.9 water-immersion lens.