Canine hair follicle samples (n=12) were collected from surgical cases brought to Madras Veterinary College Teaching Hospital. Collected skin samples were rinsed in normal saline and fixed in 10 per cent neutral buffered formalin. The fixed tissues were dehydrated in ascending grades of alcohol, cleared in xylene and embedded in paraffin wax (Bancroft and Stevens, 1990). Tissue sections were cut at 3-5µm thickness and used for the routine haematoxylin-eosin staining method.

Canine hair follicles were divided into four regions as bulb, supra bulb, isthmus and infundibulum from the base to outward. The hair follicle’s bulb and bulge regions were well demarcated by the indentation of supra bulb region. Similar to murine hair follicles, canine hair follicle also showed eight layers in their cross sections: outer root sheath (ORS), inner root sheath (IRS), henley’s layer, huxley’s layer, internal layer, cuticle, cortex, medulla from outward to inwardsin their cross section (Alcolea and Jones, 2014).

Paraffin sections of the canine skin samples were immunostained with CK15 and CK19 antibodies and location of HFSCs in the hair follicle was detected. In longitudinal sections of hair follicles, the positive CK15 (Fig. 1) and CK19 cells (Fig. 2) were observed to be located above the supra bulb region of the hair follicles and defined as bulge. In human anagen hair follicle, CK19 was expressed within the bulge and suprabulbar ORS and also intermittently between these two region in the bulge region (Purba et al., 2014).

In cross sections of hair follicles, the positively stained cells for CK15 (Fig. 3) and CK19 (Fig. 4) respectively were found to be on the periphery of the outer root sheath of the hair follicle. The cHFSCs were located on the peripheral layer of the ORS and near the insertion point of APM, as described by Ohyama et al. (2006) in human and Kobayashi et al. (2009) in canine hair follicle.

Arrector pili muscle was also immunostained with smooth muscle actin (SMA) antibody which showed the insertion point of the muscle near the hair follicle which served as a landmark to locate the cluster of bulge cells in cross section of hair follicles (Fig. 5). Diverse protrusions in the bulge region of HF were filled with more densely packed ORS cells exclusively at the insertion point of arrectorpili muscle (APM) level and positively stained with CK15 surface marker (Fig. 6).

ORS protrusions were reported in another study as follicular trochanter, observed at the point of APM insertion in humans (Tiede et al., 2007). This result also corroborates well with findings of Lyle et al. (1998) and Wang et al. (2012) in human.

In FITC conjugated Antibody staining, the CK15 positively stained cells were observed on the peripheral layer of outer root sheath of the hair follicle. The positive cells showed green fluorescent cytoplasm and blue fluorescent nucleus (Fig. 7). The bulge region of canine hair follicles were positively stained with CK19 and CK15 marker in the outermost layer of the ORS as reported by Commo et al. (2000) and Gho et al. (2004) who observed CK19 positive reaction in human HF and Kobayashi et al. (2009) reported CK15 positively in canine HF.

Immunocytochemistry of isolated and cultured cells from canine hair follicle explants revealed positive expression of CK15 with green cytoplasmic fluorescence as per Kobayashi et al. (2009) (Fig. 8). According to Kloepper et al. (2008), CK15, CK19 and CD200 were the most useful positive surface markers for human bulge cells. Similar to the human scalp as demonstrated by Inoue et al. (2009), CK15 and CK19 expression was mostly found in peri- bulge and sub-bulge areas of the canine hair follicle in this study.

We are thankful to Dean, Madras Veterinary College, Chennai-07, for providing facilities and financial support during the entire research work. We acknowledge the Department of Surgery and Radiology for providing help during sample collection and Centre for Stem Research and Regenerative Medicine, Madras Veterinary College, for providing lab facilities to carry over the work.