Micropipette

Injections were performed under a microscope with a micropipette connected to a microsyringe (Drummond Scientific Co., Broomall, PA, USA) as described (Hu et al., 2010 (link)). P2X7R antagonist BBG (0.8%) was dissolved in sterile saline and injected 5–7 days before IOP elevation. The glass pipette filled with drug was passed through the sclera at a point approximately 1 mm from the limbus into the vitreous cavity. The total volume injected was 5 μl over a 30 s time period.

Tumor-bearing mice were anesthetized with a mixture of ketamine and xylazine and i.v. injected with 2 nmols (based on fluorophore concentration) of IRDye680RD-conjugated (m)anti-MMR Nbs and IRDye680RD-conjugated (biv)anti-MMR Nbs. Blood was collected by tail vein sampling at 2.5, 5, 10, 30 and 60 min after Nb injection using a micropipette (Drummond Scientific Company). Samples were then acquired with the IVIS Lumina III system (Perkin Elmer). Images were analysed using Living Image 4.3.1 software (Perkin Elmer) and data expressed as Radiant Efficiency. Nb circulatory half-life was calculated with GraphPad, using one-phase decay equation.

All drugs used in this study were prepared on the day of experiment, backloaded to a micropipette (Drummond) and slowly injected (at 20 nl per min, total 40–50 nl) to their target cortical layer. The pipette was angled to avoid damaging the dendrites under study.
Sensory stimulation was provided by a single short electrical pulse (1 ms, 100 V) given to the contralateral hindpaw through a pair of conductive adhesive strips (Skintact).

The tear fluid was collected non-invasively during the ocular surface evaluation as reported previously^{27 (link)}. The same investigator (HM) collected the tear fluid in all cases. To avoid the effect of eye drops on the ocular surface measurements, participants were instructed not to use any eye drops on the day of tear fluid sample collection. The patient lay on his/her back on the bed and the clinician used a micropipette (Drummond Scientific, Broomall, PA, USA) to collect 2 μl of tear fluid from the external eyelid margins. The collected tear fluid was immediately stored at − 80 °C until analysis. The collected tears were measured using the BD CBA Human Anaphylatoxin Kit (BD Biosciences, San Jose, CA, USA) for CAPs (C3a, C4a, and C5a). The CAPs in the tear fluid were measured according to the previous reports and the manufacturer's instructions^{17 (link),22 (link)}.

As shown in Supplementary Fig. 1, the imaging setup consisted of a micro-periscope, an LED (peak wavelength 535 nm, Cree), an excitation filter (555/20 bandpass, AHF), an emission filter (605/55 bandpass, AHF), a dichroic mirror (cutoff wavelength: 565 nm, AHF), a 80 × 80 pixel high-speed CCD camera with frame rate of 125 Hz (Redshirt Imaging), a 10 × infinity corrected objective (58–372, Edmund Optics), and a tube lens (Optem, RL091301-1). The calcium indicator Cal-590 AM (AAT Bioquest) was backloaded into a micropipette (Drummond) and slowly injected (at 20 nl per min, total 40–50 nl) to L5 (~ 1200 μm below the pia) of the hindlimb area 1.5–2 h before imaging experiments. The pipette remained there for at least 5 min after injection. The injection pipette was angled such that spillover dye, if any, would not be loaded in neurons in upper layers of the hindlimb area. ΔF/F was calculated as (F–F₀)/F₀, where F is the fluorescence intensity at any time point and F₀ is the average intensity over the prestimulus period for 100 ms. The eYFP fluorescence upon excitation was measured before loading the calcium indicator and was subtracted from ΔF/F.