Anti iba1

L4 DRGs were dissected, post-fixed, and incubated overnight with 30% (w/v) sucrose. DRGs were embedded in optimum cutting temperature, and 20 μm sections were cut in a cryostat and processed for immunofluorescence. The primary antibodies used in this study were: anti-cfos (1:500, cat #MA1-21190, Thermo Fisher Scientific, Waltham, MA, USA) and anti-Iba 1 (1:100, cat #PA5-21274, Thermo Fisher Scientific, Waltham, MA, USA). Secondary antibodies were: Alexa Fluor 647 (1:500, cat #A32733, Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 488 (1:1000, cat #A11001, Thermo Fisher Scientific, Waltham, MA, USA. DAPI (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) was used as a nucleus marker. The coverslips were fixed on slides with Fluormount (00-4958-02, Thermo Fisher Scientific, Waltham, MA, USA). Imaging was performed using a confocal microscope (Leica TCS SP8, Leica Microsystems, Mannheim, Germany). The fluorescence intensity was quantified using LAS X Software (Leica Microsystems, Mannheim, Germany) and macrophages present in DRG were quantified by counting the total iba1 positive marked cells in the histological sections.

Cryosections were fixed in formalin for 1 h at room temperature and dehydrated in an ascending ethanol series. A multiplex was stained by blocking endogenous peroxidase with hydrogen peroxide prior to incubation with anti-iba1 (polyclonal Ab #019-19741, Wako, Fijufilm, Japan), followed by incubation with the EnVision + polymer (Agilent, USA) and the OPAL system according to manufacturer’s instructions (Akoya Biosciences, USA). This staining cycle was repeated with anti-F4/80 (clone D2S9R, Life Technologies, USA). The following OPAL dyes were used for visualization: OPAL-520 for iba1 and OPAL-620 for F4/80. A second multiplex was stained in a similar manner as described above with anti-GFAP (clone 2.2B10, Invitrogen, USA) and visualization with OPAL-540, followed by anti-iba1 and visualization with OPAL-520. Nuclei were stained using 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Sigma-Aldrich, USA), and slides were coverslipped in Fluoromount G (Southern Biotech). Multispectral images were acquired using a Vectra^® 3 imaging system (Akoya Biosciences, USA). The inForm software (version 2.4.8) was used for spectral unmixing and cell segmentation as well as cell phenotyping. The cell phenotypes were quantified using R and RStudio (version 1.3.1073).

Coronal sections of the brain were stained by standard immunohistochemistry procedures as described previously.²⁵ Briefly, the brain sections were incubated with PBS containing 0.1% Triton X‐100 and 5% goat serum (Gibco) for 30 minutes. Then, the sections were incubated overnight at 4°C with the following antibodies as appropriate: anti‐CYP4A (1:200; Abcam, ab3573), anti‐NeuN (1:200; Millipore, MAB377), anti‐GFAP (1:200; Invitrogen) and anti‐Iba1 (1:200; Invitrogen). Nuclei were labelled with DAPI (1:1000, Invitrogen). All sections were imaged using an A1 Si confocal microscope (Nikon) and analysed using ImageJ.
TUNEL staining was used to detect apoptosis and was performed according to the manufacturer's protocols (Roche). Briefly, the sections were treated with 0.3% hydrogen peroxide for 30 minutes and then incubated with proteinase K for 45 minutes. Thereafter, the sections were immersed in TUNEL reaction solution in the dark for 60 minutes. Finally, the sections were labelled using DAPI (1:1000; Invitrogen), and the ratio of TUNEL‐positive to DAPI‐stained cells was calculated to assess the degree of apoptosis.