Histoclear

THP-1 cells co-cultured in the absence or presence of Alhydrogel^® were pooled across respective cell treatments and pelleted via centrifugation at 800g for 10 min, as previously described (Mold et al. 2014 (link)). Briefly, cells were re-suspended in 4% w/v paraformaldehyde in 25 mM PIPES, pH 7.4 for 20 min. Fixed cell treatments were washed three times with a 50 mM PIPES buffer adjusted to pH 7.4 with sodium hydroxide pearls, of which 30 μL 5% w/v molten agar was added to the final pellets. HPLC-grade ethanol was used in all procedures whenever required. Agar-cell blocks were gradually dehydrated through an ethanol gradient of 30, 50, 70, 90, 98 and 100% v/v for 20 min, followed by incubation in fresh 100% v/v ethanol, for a further 20 min. Blocks were cleared via transfer into Histo-Clear™ (National Diagnostics, Nottingham, UK) for 40 min, with one change of fresh Histo-Clear™ halfway through. Cleared agar-cell blocks were infiltrated in molten paraffin wax at 60 °C for 40 min and rapidly cast onto Tissue-Tek embedding cassettes (VWR, Sakura^® Finetek, Harrisburg, PA, US) on ice, followed by overnight incubation at 4 °C to set the final paraffin-embedded blocks.

In vitro models were washed in PBS prior to fixation in 4% paraformaldehyde (Fisher) for 2 h. Samples were dehydrated through a series of ethanols, followed by incubation in Histoclear (National Diagnostics, United States) then in 1:1 Histoclear:wax. Models were further incubated in wax before embedding and sectioning.
Paraffin sections were deparaffinized in Histoclear and rehydrated to dH₂O before being stained in Mayer’s Hematoxylin (Sigma-Aldrich) for 5 min. Slides were then washed in dH₂O and submerged in alkaline EtOH to blue the nuclei. Samples were dehydrated to 95% EtOH counter-stained in Eosin followed by dehydration to 100% EtOH. Slides were cleared twice in Histoclear and mounted in Omni-mount (National Diagnositcs) before imaging on a Leica microscope.

IHC staining was performed in formalin-fixed, paraffin-embedded (FFPE) aortic samples. Human tonsil sections were used as a positive control. Briefly, 4 µm sections were deparaffinized in Histoclear (National Diagnostics) and then dehydrated through graduated alcohols. Antigen retrieval was performed in R-Universal epitope recovery buffer (Aptum Biologics Ltd #AP0530-125) using 2100 Retriever (Aptum Biologics Ltd). EnVision^TM+Dual Link system (Dako) was used for chromogenic detection of the primary antibodies. Sections were counterstained with hematoxylin (Sigma-Aldrich), rehydrated through graduated alcohols, cleared in Histoclear, and then mounted with Histomount (National Diagnostics).

Whole-mount hearts were fixed in 4% paraformaldehyde (PFA) (Sigma, 158127) for 15-30 minutes. For tissue sections, hearts were fixed in 4% PFA for 15-30 minutes (for cryosections) or overnight (for paraffin sections). All specimens were subsequently thoroughly washed with PBS to remove excess PFA. To prepare samples for paraffin embedding, samples were dehydrated through an increasing ethanol concentration series (50/70/85/95/100% (v/v) ethanol in PBS), washed twice with 100% Histo-Clear (National Diagnostics, HS-200), and then moved to 50/50 Histo-Clear/paraffin at 65°C. Samples were then washed with at least three changes of 100% paraffin (RA Lamb, 12624077). All incubations were for 30-60 minutes. For cryo-embedding, samples were first incubated overnight at 4°C in 30% (w/v) sucrose (Fisher, S/8600/53), The following day, samples were incubated in 50/50 30% sucrose/optical cutting temperature compound (OCT) (CellPath, KMA-0100-00A) for 30 minutes, followed by 3 washes in 100% OCT; samples were then snap-frozen in 100% OCT.