to induce nafld and nash, mice were fed a western diet ( wd ) high in fat, sugar and cholesterol for 24 or 36 weeks as described previously ( remmerie et al., 2020 ). this consisted of 58 % fat, 1 % cholesterol ( research diets; d09061703i ) and drinking water was supplemented with 23. 1g / l fructose ( mpbio ) and 18. 9g / l sucrose ( vwr ). control mice were fed a standard diet with 11kcal % fat with corn starch ( d12328i; research diets ). cells were pre - incubated with 2. 4g2 antibod y ( bioceros ) to block fc receptors and stained with appropriate antibod ies at 4°c in the dark for 30 - 45 minutes. cell viability was assessed using fixable viability dyes ( efluor780 or efluor506; thermo fischer ) and cell suspensions were analyzed with a bd facsymphony or purified using a bd symphony s6, bd facsaria ii or iii. nuclei were sorted on basis of dapi positivity and size. analysis was performed with flowjo software ( bd ). intracellular staining for cd207 was performed by fixing and permeabilizing extracellularly stained cells according to the manufacturer ’ s instructions using the foxp3 fixation / permeabilization kit ( thermo fischer ). confocal staining was performed as described previously ( bonnardel et al., 2019 ). immediately after sacrificing mice with co 2, inferior vena cava were cannulated and livers were perfused ( 4 ml / min ) with antigenfix ( diapath ) for 5 min at room temperature. after excision, 2 - 3 mm slices of livers were fixed further by immersion in antigenfix for 1h at 4°c, washed in pbs, infused overnight in 34 % sucrose and frozen in tissue - tek oct compound ( sakura finetek ). 20 μm - thick slices cut on a cryostat ( microm hm 560, thermo scientific ) were rehydrated in pbs for 5 min, permeabilized with 0, 5 % saponin and non - specific binding sites were blocked for 30 min with 2 % bovine serum albumin, 1 % fetal calf serum and 1 % donkey or goat serum for 30 minutes. tissue sections were labeled overnight at 4°c with primary antibod ies followed by incubation for 1h at room temperature with secondary antibod ies. when two rat antibod ies were used on the same section, the directly conjugated rat antibod y was incubated for 1h after staining with the unconjugated and anti - rat secondary and after an additional blocking step with 1 % rat serum for 30 minutes. slides were mounted in prolong diamond, imaged with a zeiss lsm780 confocal microscope ( carl zeiss, oberkochen, germany ) with spectral detector and using spectral unmixing and analyzed using imagej and qupath software. experiments were performed using the rnascope multiplex fluorescent v2 assay kit ( acdbio 323100 ). probes targeting intronic regions for hs - cd5l ( acdbio 850511 ), mfa - cd5l ( acdbio 873211 ), mm - cd5l ( acdbio 573271 ), mm - flt3 ( acdbio 487861 ), mm - xcr1 ( acdbio 562371 ), mm - mafb ( acdbio 438531 ) and mm - mgl2 - o1 ( acdbio 822901 ) were custom - designed and synthesized. they were then labelled with tsa opal 520 ( perkinelmer fp1487001kt ), tsa opal 540 ( perkinelmer fp1494001kt ), tsa opal 570 ( perkinelmer fp1488001kt ), tsa opal 620 ( perkinelmer fp1495001kt ) or tsa opal 650 ( perkinelmer fp1496001kt ). tissues were fixed for 16 hours in antigenfix ( diapath p0016 ), dehydrate d and embedded in oct as described above. slices were pre - treated with hydrogen peroxide for 10 min and protease iii for 20 min. the recommended antigen retrieval step was not performed in order to preserve epitope integrity. probes were hybri diz ed and amplified according to the manufacturer ’ s instructions. slides were then stained for protein markers as described above. mice were euthanized by means of carbon dioxide ( co 2 ) overdose. the liver was excised and consequently trimmed, on ice, to smaller tissue pieces fitting the 10x visium capture area. trimmed tissue pieces were embedded in tissue - tek® o. c. t. ™ compound ( sakura ) and snap frozen in isopentane ( sigma ) chilled by liquid nitrogen. embedded tissue pieces where stored at - 80°c until cryosectioning. a 10x visium spatial gene expression slide was placed in the cryostat ( cryostar nx70 thermo fisher ) 30 minutes prior to cutting. 10 μm sections where cut and placed within the capture area. single 10x visium spatial gene expression slides were stored in an airtight container at - 80°c until further processing. 10x visium cdna libraries were generated according the manufacturer ’ s instructions. in short: tissue sections where fixed in chilled methanol. a h & e staining was performed to assess tissue morphology and quality. tissue was lysed and reverse transcription was performed followed by second strand synthesis and cdna denaturation. cdna was transferred to a pcr tube and concentration was determined by qpcr. spatially barcoded, full length cdna was amplified by pcr. indexed sequencing libraries where generated via end repair, a - tailing, adaptor ligation and sample index pcr. full length cdna and indexed sequencing libraries were analyzed using the qubit 4 fluorometer ( thermo fisher ) and agilent 2100 bioanalyzer. liver slices were prepared as described above for the classical visium protocol. slices were dried for 1 min at 37°c and subsequently fixed using 1 % paraformaldehyde in pbs. next, slices were blocked for 30 min ( 2 % bsa, 0. 1ug / ul salmon sperm, 0. 5 % saponin, 1 u / μl protector rnase inhibitor ( roche ) in 3x ssc ) and incubated with the oligo - conjugated antibod y staining mix ( 2 % bsa, 0. 1μg / μl salmon sperm, 0. 5 % saponin, 1 u / μl protector rnase inhibitor, 10um polyt - blocking oligo ( ttttttttttttttttttttttt t t / 3invdt / ), in 3x ssc ) for 1h at 4°c. slides were mounted ( 90 % glycerol, 1 u / μl protector rnase inhibitor ) and imaged on zeiss axioscan z1 at 20x magnification. samples were then processed for a transcriptomic experiment as per manufacturer ’ s instructions ( visium, 10x genomics ) with modifications to also capture antibod y tags. in short, tissue was permeabilized using tissue removal enzyme ( tissue optimization kit, 10x genomics ) for 9 minutes, as determined by a tissue optimization experiment ( 10x genomics, visium spatial tissue optimization ). after reverse transcription, 2 μl of 100 μm fb additive primer ( ccttggcacccgagaatt c c a ) per sample was added to the second strand synthesis mix. during cdna amplification 1 μl of 0, 2 μm fb additive primer ( ccttggcacccgagaatt c c a ) was added. after cdna amplification, antibod y products and mrna derived cdna were separated by 0. 6x spri select. the purified full - length cdna fraction was quantified by qrt - pcr using kapa sybr fast - qpcr kit on a pcr amplification and detection instrument. after enzymatic fragmentation indexed sequencing libraries were generated via end repair, a - tailing, adaptor ligation and sample index pcr. the supernatant containing antibod y product was cleaned up by two rounds of 1. 9x spri select. next, 45 μl of the purified antibod y fraction was amplified with a 96 deep well reaction module: 95°c for 3 min; cycled 8 times: 95°c for 20 s, 60°c for 30 s, and 72°c for 20 s; 72°c for 5 min; end at 4°c. adt libraries were purified once more with 1. 6x spri select. full length cdna, indexed cdna libraries and antibod y libraries were analyzed using the qubit 4 fluorometer ( thermo fisher ) and agilent 2100 bioanalyzer. the separation of the cdna and adt libraries were performed according to the manufacturer ’ s instructions ( 10x genomics ). the macsima™ imaging system is a fully automate d instrument combining liquid handling with widefield microscopy for cyclic immunoflu orescence imaging. in brief, staining cycles consisted of the following automate d steps: immunoflu orescent staining, sample washing, multi - field imaging, and signal erasure ( photobleaching or realease ). cryosectioned slices on slides were taken out of the - 80°c storage and the appropriate macswell™ imaging frame was mounted immediately on the slide.