Tissue tek oct freezing media

LNs were excised from naïve and infected animals, fixed overnight with 4% paraformaldehyde/PBS followed by dehydration in 30% sucrose/PBS prior to embedding in Tissue-Tek OCT freezing media (Sakura Finetek). 16 to 20 micron-thick sections were cut on a HM 550 cryostat (Thermo Scientific) and adhered to Superfrost Plus slides (VWR). Sections were permeabilized and blocked in PBS containing 0.3% Triton X–100 (Sigma) and 10% goat serum (Jackson Immunoresearch). This was followed by incubation with AlexaFluor 647-conjugated rat anti-CD3 (17A2) (BD Biosciences). Incubation with unconjugated rat anti-PNAd (MECA–79) (BD Biosciences) was followed by staining with AlexaFlour 647-conjugated secondary antibodies (Invitrogen). Slides were mounted with Prolong Gold (Invitrogen). 3D image stacks of LN sections were acquired on a LSM 780 confocal microscope (Carl Zeiss MicroImaging). Images are displayed as 2D maximum intensity projections using Imaris software (Bitplane).

Ears infected with BCG-RFP were fixed overnight with 4% paraformaldehyde/PBS followed by dehydration in 30% sucrose/PBS before embedding in Tissue-Tek OCT freezing media (Sakura Finetek). A total of 16- to 20-μm-thick sections were cut on a Microm HM 560 cryostat (Thermo Scientific) and adhered to Superfrost Plus slides (VWR). Sections were permeabilized and blocked in PBS containing 0.3% Triton X-100 (Sigma) and 10% goat serum (Jackson Immunoresearch). This was followed by incubation with polyclonal rabbit anti–mPGES-1 (Cayman Chemicals) and staining with Alexa Flour 488–conjugated goat anti-rabbit secondary Ab (Invitrogen). Slides were counterstained with Hoechst and mounted with Prolong Gold (both Invitrogen). 3D image stacks were acquired on a LSM 800-Airy confocal microscope (Carl Zeiss MicroImaging). Images are displayed as 2D maximum intensity projections using Fiji software (ImageJ) (24 (link)).