Uplsapo objective

An Abberior STED/RESOLF system (Abberior Instruments GmbH, Germany) equipped with a 100 × oil-immersion Olympus UPLSAPO objective (NA 1.4, working distance 0.13 mm) was used. Abberior-FIP-IgM was excited with a pulsed 635 nm laser and depleted using a 775 nm pulsed depletion laser, and emission signal was detected in the Cy5 channel (685/35 nm). Confocal images of Alexa Fluor® 488 anti-IgM F(ab)’₂ were taken using the 488 nm laser, and the emission signal was detected in the GFP channel (525/25 nm). A 20 nm × 20 nm pixel size was used.

Primary antibodies used for Western blottings were as follows: rat anti-HA (Roche catalog 11-867-423), mouse anti-RPS5 (Abcam, ab58345), rabbit anti-RPS18 (Abcam, ab91293), rabbit anti-RPS15 (Abcam, ab90902), rabbit anti-RPL11 (Abcam, ab79352), rabbit anti-TUJ1 (Covance catalog MRB₄₃₅P), mouse anti-Rpl22 (Santa Cruz Biotechnology, sc-373993), rabbit anti-gERK (Sigma, M5670), and rabbit anti-albumin (Cedarlane, CLAG5140).
Primary antibodies used for immunostaining were as follows: rat anti-HA (Roche catalog 11-867-423), mouse Y10B (Abcam catalog ab37144), rabbit anti-TUJ1 (Covance catalog MRB₄₃₅P), rat anti-MBP (Millipore, MAB395-1ML), and chicken anti-NFH (Abcam, ab72996). Fluorescent secondary antibodies were from Jackson ImmunoResearch. Staining was observed using an Olympus FV1000 Confocal laser-scanning microscope at 40× or 60× magnification with oil-immersion Olympus UPLSAPO objective and was analyzed with FV10-ASW2.0 software.

Confocal images at the Olympus FV1000 equipped with a 60×/1.35 numerical aperture Oil UPLSApo objective (Olympus, Hamburg, Germany) and a temperature controlled incubation chamber (EMBL, Heidelberg, Germany) were acquired in sequential mode frame by frame with 2× line averaging. The pinhole was set to 2.5 airy units. mCitrine was excited with a 488 nm Argon laser (GLG 3135, Showa Optronics, Tokyo, Japan), mCherry/Atto590 with a 561 nm DPPS laser (85-YCA-020-230, Melles Griot, Bensheim, Germany), and Alexa647 with a 633 HeNe laser (05LHP-991, Melles Griot, Bensheim, Germany) using a DM405/488/561/633 dichroic mirror. mCitrine fluorescence was detected between 498 and 551 nm using the SDM560 beam splitter. Atto590/mCherry fluorescence was detected in the bandwidth of 575–675 nm and Alexa647 fluorescence between 643 and 743 nm. With these settings, no bleed through between mCitrine and Atto590 or mCherry was observed. Live cells were imaged in imaging media at 37 °C and 5% CO₂ and stimulated with 20 ng ml⁻¹ EGF, 100 ng ml⁻¹ EGF, or 0.33 mM PV.

Seven days after mSNIs or sham surgeries, the lumbar lymph nodes (LLNs) of adult rats (n = 5 animals per group) were removed to determine reactive hypertrophy and relative weights of those pooled peripheral lymphoid organs to their corresponding body weights as described previously [18 (link)]. At the same time points, the LLNs of adult rats (n = 5 animals per group) were longitudinally sectioned on a cryostat in 10-μm thickness and processed for chromogenic IHC as described previously [18 (link)]. In brief, slide-mounted frozen sections of LLNs were incubated overnight at 4 °C with the following PAbs: mouse monoclonal anti-αβTCR (1:1000), mouse monoclonal anti-CD4 (1:1000), and rabbit polyclonal anti-IFNγ (1:100, BioLegend, 507801; San Diego, CA, USA). The positive cells in the paracortical zones (PCZs) of these lymph nodes were imaged with a × 20 (NA = 0.75) Olympus UPLSAPO objective (at least three fields/section, at least eight sections/sample).