Alexafluor 488 anti mouse igg secondary antibody

Lumbrical and flexor digitorum brevis (FDB) muscles located in the hindlimb paws of mice were dissected and stained, as previously described^{28 (link),84 (link)}. Briefly, after dissection, tissues were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 8–10 min in phosphate buffered saline (PBS) then permeabilized with 2% PBS-Triton X-100 for 30 min to increase antibody penetration. Then, blocking solution (5% donkey serum in 0.05% PBS-Triton X-100) was added to the tissue for 1 h followed by multiple washes of 15–20 min each. Overnight incubations with mouse anti-synaptic vesicle 2 (SV2, 1/20, DHSB) and mouse anti-neurofilament medium chain (2H3 1/50, DSHB) in blocking solution at 4 °C were performed to visualise the pre-synaptic NMJ component. After overnight incubation, multiple washes were conducted with PBS-1X for at least 1 h. AlexaFluor-488 anti-mouse IgG secondary antibody (1/2000, Invitrogen) was added together with labelled α-bungarotoxin (to visualise post-synaptic acetylcholine receptors) in blocking solution for 1.5 h at room temperature (RT) (α-btx, Life Technologies). Then, muscles were washed several times and whole-mounted for imaging.

We used a similar method to monitor the upper genital tract infection in mice as described elsewhere [31 (link)]. To recover viable Chlamydia from the UGT of C. muridarum infected wild-type and TLR3-deficient mice, each mouse’s UGT tissue sample was homogenized in 300μl of SPG using a 2-ml Dounce tissue grinder (Sigma) and subsequently passaged through a 20-gauge syringe. After a brief sonication, the released chlamydial EBs were titrated on McCoy cell monolayers as described previously in [8 (link), 28 (link), 30 (link)]. Briefly, chlamydial elementary bodies harvested from infected tissue were serial diluted 1:10 in SPG and quantified as inclusion forming units (IFUs) per milliliter on McCoy cell monolayers. The total number of IFUs per ml was calculated based on the enumeration of fluorescent inclusion bodies in the cells whereby antibody specific for chlamydial LPS was used to detect chlamydial inclusions in the infected McCoy cells. Detection of the chlamydial LPS was done via Alexa Fluor 488 anti-mouse IgG secondary antibody (Invitrogen/Life Technologies; Carlsbad, CA), and immuno-staining results were scanned and recorded by EVOS imaging system (Thermo-Fisher, Pittsburgh, PA).