No antibody used in this study is species specific for the dog, but they still can be used due to interspecies cross-reactivity. All antibodies have been used previously in multiple other canine studies [20–26].
Variation in vimentin and desmin staining pattern was widely present. This might be due to the varying epitope sensibility, caused by the intrinsic patient material variability regarding time of postmortal sampling and fixation, the age of the paraffin blocks, and age, sex and breed variation of the animals. However, the used material reflects similar variability in intended patient populations to be studied for spontaneously occurring hepatic fibrosis, and thus provides useful insight in normal baseline variation.
In the formalin fixed paraffin embedded normal canine liver vimentin staining did not differentiate between fibroblasts and MF in the portal area and the perivenous stromal tissue. Moreover, HSC stained generally negative. Therefore, we conclude that vimentin antibody is not useful in paraffin sections as a marker for canine portal MF or HSC. Desmin stained MF in the portal area, around the sublobular hepatic vein and in Glisson's capsule. HSC stained inconsistently, with large variation between slides, so we conclude that desmin is not a sensitive marker for canine HSC. This is in contrast to man , but in accordance to rat .
In our laboratory, both α-SMA and HHF35 do identify myoepithelial cells in canine mammary gland. These monoclonal antibodies recognise different epitopes: a NH2 terminal decapeptide (α-SMA), and α and γ muscle actin (HHF35). The chance of formalin-induced epitope masking was regarded smaller by use of two different monoclonal antibodies for the same peptide. Therefore, both markers were investigated in related (regarding possible contractility) cells in the liver, being HSC and portal MF. In formalin fixed paraffin sections these cells can be easily identified in the normal canine liver by immunohistochemical staining for both α-SMA and HHF35. Both antibodies produced almost identical results and stained both solitary MF in the portal areas as well as HSC in the hepatic parenchyma. The presence of small lipid vacuoles in positively staining perisinusoidal cells as well as the TEM immunohistochemical results confirms the nature of the latter cells as HSC. The vitamin A-storing HSC usually stained positive for α-SMA but reacted only rarely to HHF35, suggesting differentiation in staining characteristics between less contractile vitamin A-storing HSC and more contractile HSC. The present finding of α-SMA reactivity which was diffusely present throughout the hepatic parenchyma in the normal canine liver is in contrast with findings in normal human and rat liver, where the majority of hepatic lobules are devoid of α-SMA positive HSC, or only show weak positivity [2, 6, 9, 12]. In our opinion, this indicates a species-specific expression pattern for the dog. Rat and human HSC upregulate α-SMA expression upon activation [2, 6, 28] and this positive staining of activated HSC is considered to represent increased contractility of the cells [29, 30].
Debate still exists regarding the contribution of non-activated quiescent HSC to sinusoidal blood flow and blood pressure in man and rat [9, 11]. Our findings of α-SMA staining in HSC of normal dogs may suggest a more active role in controlling microvascular blood flow compared to the rat. Interestingly, the dog is unique in that it has a spirally shaped smooth muscle surrounding the sublobular hepatic veins consistent with a more active regulation of the hepatic blood flow in the dog compared to other species .
Despite positive staining of HSC for α-SMA in normal dogs reflecting contractility we feel it appropriate to regard these cells as "quiescent" HSC. This is in line with other species as HSC are most likely not activated in the sense of enhanced matrix- or TGF-β production. In the dog discrimination between quiescent and activated HSC does not seem possible with antibodies directed against α-SMA and HHF35. However, morphological changes or functional changes such as increased cell size, loss of lipid vacuoles and enhanced production of TGF-β and other substances may be helpful.
The absence of reactivity of portal MF and HSC in the normal canine liver to synaptophysin and GFAP indicates that in contrast to man and rat [2, 3, 6], canine portal MF and HSC do not seem to express the used markers in the normal liver. Moreover, in frozen sections from a dog with chronic active hepatitis which likely contained activated MF and HSC, these cells did also not react to antibodies for neural crest markers NCAM, GFAP and synaptophysin. As in frozen samples antigens retrieval is not necessary, we conclude that not only in the quiescent state but also in the activated state, canine portal MF and HSC do not bind the used antibodies for NCAM, GFAP and synaptophysin. Although probably limited by species-specificity of the antibodies, this study could be expanded by the use of more frozen tissue samples, or by the use of other potential markers, reacting positively on hepatic MF in other species, like NGF, BDNF, NT-3, NCAM , Foxf1  or fibulin-2 .