Eclipse microscope

The specimen was documented on an upright Nikon Eclipse microscope with transmitted light. Objective lenses of 10×, 20×and 40×magnifications were used resulting in overall magnifications of 100×, 200× and 400×. Due to the specific optical properties of the chert resulting in a very limited depth of field, the specimen was documented with stacks, in higher magnifications with two adjacent stacks. Classical image fusion of these stacks does not yield sufficient results. We used the techniques described in Haug et al. (2009) (link) and Haug et al. (2012) for projecting the stacks in 3D. Stereo images were used to present these projections. The stack could be turned 180° to allow a view on the other side (factually simple depth inversion). Interpreted structures are presented as color markings alongside the original projection. Based on these, simplified 3D models were produced in Blender.
The modern specimen of S. coleoptrata was documented on a Nikon Eclipse microscope exploiting the autofluorescence of the specimen. Workflow followed Haug et al. (2011) (link). Mouth parts were sequentially removed to reveal the arrangement of underlying ones.

C57BL/6 mice were pretreated with RRx-001 to establish an in vivo model of neuroinflammation. Seven days after LPS injection, the mice were perfused with precooled PBS and then 4% paraformaldehyde and their brains were taken and immersed in 4% paraformaldehyde for 3 days at 4°C. The brains were then dehydrated at 4°C for 3 days in 20 and 30% sucrose, respectively. After being frozen overnight at −80°C, 20 µM-thick slices of the midbrain were obtained at −20°C by using a CM1950 Freezing Microtome (Leica, Wetzlar, Germany). Microglia and astrocytes were visualized by immunohistochemical staining with anti-Iba1 antibody (Wako Chemicals, Tokyo, Japan) and anti-GFAP antibody (Sigma-Aldrich, St. Louis, United States), respectively. The dopaminergic neurons were shown by immunohistochemical staining with anti-tyrosine hydroxylase (TH) antibody (Millipore, Billerica, United States). The nuclei were stained with DAPI. Finally, the slices were imaged by using an inverted Nikon Eclipse Microscope (Nikon, Tokyo, Japan) and the fluorescence intensity was analyzed by using ImageJ software.

Tumor xenograft cryo-sections were fixed in 4% PFA, treated with 0.4% pepsin in 0.2 N HCl, 3% H₂O₂–PBS, and then permeabilized and assayed for WT-1 (Dako) expression with anti-mouse EnVision-HRP (Dako). Calretinin antibody (Dako) was used on sections fixed in 4% PFA, followed by heat-induced antigen retrieval and treatment with 0.3% H₂O₂–PBS and permeabilization. Detection was performed using streptavidin/horseradish peroxidase (Dako). Mesothelin staining was assessed using a secondary antibody conjugated with a green fluorescent dye. For D2-40 staining (Dako), heat antigen retrieval was performed using Target Retrieval Solution S1700 (Dako) and permeabilization in PBS 10% NGS 0.3% Triton X-100. Mouse IgG and the omission of primary antibody were used as negative controls [25 (link)]. Digital images were captured by a Nikon Eclipse microscope (Nikon Europe).

For transplantation, mammary glands containing PN1a tissue were removed from donor mice at 8 weeks post-transplantation, minced into 1 mm fractions with a scalpel and re-transplanted into the cleared fat pads of 3 week-old female Balb/c mice as previously described (18 (link)). At 8 or 16 weeks post-transplantation, inguinal mammary glands containing PN1a outgrowths were fixed in cold 4% paraformaldehyde for 2 h and stained with carmine alum overnight (six mammary glands per timepoint). The next day, glands were dehydrated and imaged on a Leica M165 FC stereoscope (Leica Biosystems) as previously described (19 (link)). After imaging, mammary glands were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) as previously described (20 (link)). H&E images were captured using an upright Nikon Eclipse microscope (Nikon Instruments). For tumors, mice were palpated twice weekly until tumors were measurable, and then measured three times a week. When tumors reached 1.2 cm in size, mice were euthanized and excised tumors were fixed with 4% PFA overnight and embedded in paraffin for subsequent immunostaining.