Axioplan 2 epifluorescence microscope

Cells that were TE were distinguished from cells in the ICM based on nuclear labeling for CDX2 using procedures similar to those previously published [4 (link), 25 (link), 44 (link), 45 (link)]. The primary antibody was ready-to-use mouse monoclonal antibody against CDX2 (BioGenex, Fremont, CA, USA; reference AM392-5 M, lot AM3920917) For randomly-selected embryos, mouse 1gG (1 μg/mL) was used as a negative control. The second antibody was 1 μg/ml fluorescein isothiocyanate labeled goat anti-mouse IgG. Nuclei were labeled with 1 μg/ml Hoechst 33342. Fluorescence was observed with a Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Gottingen, Germany) with a 40x objective and using Zeiss filter sets 02 (blue) and 03 (green). Digital images were acquired using AxioVision software (Zeiss) and a high-resolution black and white Zeiss AxioCam MRm digital camera. Total cell number was determined by counting nuclei labeled with Hoescht 33,342, number of TE cells was determined by counting nuclei positive for CDX2 and number of ICM cells was determined by subtracting number of TE cells from total cell number.

The probe used in this study, MTC849 probe (5′-CGTTAGCTCCACCACTAAG-3′), was designed with ARB [35 ] to target the 16S rRNA gene sequences of the symbiotic MOX of both Campeche sponge species. This probe is a modification of MTC850 probe, designed to target Marine Methylotrophic Group (MMG) 2 MOX [36 (link)]. Apart from the symbiotic and MMG2 MOX, the MTC849 probe targets the closely related Methylomonas and Methylomarinum clades. The MTC849 oligonucleotide was double-labeled with Atto594 dye (Biomers, Ulm, Germany), and applied to 8 μm sections of sponge tissue using hybridization buffer with 20% formamide as described previously [37 (link)]. These hybridization conditions are assumed to ensure specificity, given the three mismatches that the MTC849 probe had to the 16S rRNA gene sequences of all other Campeche sponge bacteria [38 (link)].The general bacterial probe EUB338 [39 (link)] was used as a positive control and the NON338 probe was used as a control for background autofluorescence [40 (link)]. Photomicrographs were acquired with a Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Jena, Germany) or with a confocal laser-scanning microscope (LSM 780, Carl Zeiss, Germany). Brightness and contrast of the images were adjusted with Adobe Photoshop (Adobe Systems, Inc., USA).

Fluorescence images of cultured cells seeded in black, clear bottom 96 well
microplates (Greiner) were acquired on a Zeiss Observer.Z1 inverted
epifluorescence microscope (Carl Zeiss, Jena) equipped with an AxioCamMR3 camera
using a 40x objective. Fluorescence microscopy of lung sections was performed on
a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Jena) equipped with
an ApoTome optical sectioning module using a 10x objective. Images were recorded
with an AxioCamMR camera (Carl Zeiss, Jena).

Fluorescence micrographs of stained splenic sections were obtained with a Zeiss Axioplan2 epifluorescence microscope (Carl Zeiss, White Plains, NY) with Chroma single-channel ET filter sets, 10x and 20x Plan-Apochromat objectives (NA 0.45 and 0.8, respectively) and a Coolsnap HQ camera controlled by MetaMorph 7.8 (Molecular Devices, Sunnyvale, CA USA). To identify OTII T cell and splenic CD11c⁺ DC locations in splenic sections, 10-μm-thick z-stacks were collected at 0.2-μm intervals, the contrast and brightness of each stained test and isotype control mAb were adjusted using Image J software in a blinded fashion. Representative compensated images were reconstructed into 3-dimension projections using MetaMorph software v7.8. Quantifying the distances in μm between transferred CFSE⁺ T cells and endogenous CD11c⁺ DCs in the T cell zones of splenic white pulp was performed using IMARIS software. IMARIS masking was applied to identify CFSE⁺ T cells and to measure the distance to the nearest CD11c⁺ DCs. Representative masked images for each strain of mice are presented in S4 Fig.