Wheat germ agglutinin alexa fluor 594 conjugate

McCoy cells seeded onto glass coverslips in 12 well trays were infected with
C. muridarum P+ and plasmid-free
C.muridarum P- (at an MOI=1.0) as described above, and then fixed at 4-hour time intervals between 0 and 40 hours post-infection using 4% paraformaldehyde for 15 minutes. Cells were washed in PBS and permeabilised in saponin buffer (0.1% saponin, 10% foetal calf serum, 0.1% sodium azide) for 1 hour at 4°C. Primary and secondary antibodies were added to the coverslips, and incubated in saponin buffer for 1 hour at room temperature; the coverslips were washed in saponin buffer between steps. A mouse monoclonal primary antibody raised against genus-specific LPS (Chlamydia Biobank Cat. No. #CT601 RRID: AB2721933) was diluted 1:1,000 and combined with an anti-mouse-Alexa Fluor® 488 conjugate secondary antibody (Invitrogen™ Cat. No. A11001 RRID AB_2534069) was used (1:200 dilution) to visualize
C. muridarum. Cells were counterstained with 1 µg/ml DAPI (Fisher Scientific) and Wheat Germ Agglutinin Alexa Fluor® 594 conjugate (Invitrogen™ Cat. No W11262)), washed a final time in PBS and mounted onto slides with Mowiol 4-88 (Sigma). Images were captured using a Leica TCP SP5 confocal microscope.

Hearts were dissected in PBS, incubated in 30% sucrose overnight at 4°C and embedded in O.C.T. Compound (Tissue-Tek) for cryosectioning. Then, 4 µm sections were stained with Wheat Germ Agglutinin, Alexa Fluor 594 Conjugate (Invitrogen, W11262) for 10 min and washed 3× for 10 min each with PBS. Sections were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and imaged under a Nikon Eclipse Ni microscope. Cardiomyocyte cell surface area was quantified using ImageJ from images of the right and left ventricles. Fifty cardiomyocytes in three view fields of three different sections from each of six hearts per genotype were measured.

12-well trays containing glass microscope coverslips were seeded with McCoy cells the day before the start of the experiment. Cells were infected with the appropriate amount of C. trachomatis A2497WT, A2497P-, or A2497P-/pSW2::GFP inoculum to give an MOI of 1. The 12 well trays were centrifuged and incubated at 37°C/5% CO₂ as described above. At each time point, cells were fixed using 4% paraformaldehyde for 15 min. Cells were washed in PBS and permeabilised in saponin buffer (0.1% saponin, 10% foetal calf serum, 0.1% sodium azide) for 1 h at 4°C. Monoclonal antibody MAb29 was added (in saponin buffer) at 1:1,000 dilution and cells were incubated for 1 h at room temperature. These were then washed three times in saponin buffer before the anti-mouse-Alexa-fluor 488 conjugate antibody (Fisher Scientific) was added at 1:200 dilution. This was again incubated for 1 h at room temperature, washed as before and counterstained with 1 μg/ml DAPI (Fisher Scientific) and Wheat Germ Agglutinin Alexa Fluor® 594 conjugate (Invitrogen), washed a final time in PBS and mounted onto slides with Mowiol mounting medium (Sigma Aldrich). Images were captured using a Leica TCP SP8 confocal microscope. Areas of inclusions were measured at each time point using ImageJ (version 1.51j8) and compared using a one-way ANOVA using GraphPad Prism (version 7.03).

Cells were collected, washed twice with PBS and fixed in 4% paraformaldehyde for 20 min. After the washing with PBS, cells were incubated in 1% BSA in PBS for 1 h and subsequently incubated with CRT (Enzo Life Sciences, 1:100), HSP70 (R&D, 1:100) or HSP90 (Enzo Life Sciences, 1:100) antibody in PBS overnight at 4 °C. Cells were washed with PBS and incubated with AlexaFluor488 goat anti-mouse (Molecular Probes, 1:200) IgG1 (CRT and HSP90) or with AlexaFluor488 goat anti-mouse IgG2A (HSP70) secondary antibody (Molecular Probes, diluted 1:200) for 1 h at 4 °C. Cells were washed with PBS and incubated with Wheat Germ Agglutinin, Alexa Fluor 594 Conjugate (Invitrogen, 5 μg/ml) for 10 min at room temperature. Then the cells were washed with PBS and mounted on slides with ProLong Gold antifade reagent with DAPI (Molecular Probes) using StatSpin Cytofuge. Cells were analysed under a DMI 6000 inverted Leica TCS AOBS SP5 tandem scanning confocal microscope and an × 63 oil immersion objective.