. Author manuscript; available in PMC: 2012 Sep 1.

Published in final edited form as: J Invest Dermatol. 2011 Nov 24;132(3 0 1):642–649. doi: 10.1038/jid.2011.377

Table 1.

Relative proportions of leukocytes (% of area)¹ in feather tips² from SL chickens with and without SLV

	Vitiligo state³

	NV	EV	AV	CV

T cells (alpha-beta+ TCR)	0.70 ± 0.17^b,⁴	3.95 ± 1.57^ab	4.92 ± 1.10^a	2.15 ± 0.67^ab
T cells (gamma-delta+ TCR)	0.47 ± 0.12	2.08 ± 1.13	2.10 ± 0.57	1.25 ± 0.35
CD4+ lymphocytes	0.61 ± 0.30^b,	2.35 ± 0.67^b	4.81 ± 0.48^a	1.49 ± 0.51^b
CD8+ lymphocytes	1.17 ± 0.52^b	3.51 ± 0.88^b	5.71 ± 0.64^a	2.37 ± 0.60^b
CD4+:CD8+	0.48 ± 0.06^b	0.68 ± 0.08^ab	0.87 ± 0.10^a	0.68 ± 0.13^ab
B cells (Bu-1+)	0.25 ± 0.15^b	2.37 ± 0.73^b	5.07 ± 1.21^a	1.53 ± 0.46^b
IgM+ cells	0.31 ± 0.05^b	1.89 ± 0.62^ab	4.30 ± 1.15^a	1.71 ± 0.48^b
T:B⁵	5.16 ± 2.66^a	2.69 ± 1.10^ab	1.52 ± 0.21^b	2.94 ± 0.69^ab

leukocyte types were determined by indirect immunoperoxidase staining. Primary unlabeled antibodies were mouse-anti-chicken monoclonal antibodies to alpha-beta T cell receptor (TCR) (cocktail of alpha-beta1 and alpha-beta 2 TCR), gamma-delta TCR, CD4, CD8, Bu-1 (B cell marker) and IgM. Binding of primary antibodies was detected by biotinylated horse-anti-mouse IgG (H+L) with avidin-biotin peroxidase complex reagents and using DAB as the substrate. Image analysis was carried out at 40× magnification under a bright field microscope. The amount of stained cells in feather tip sections was expressed as the percentage of the whole area analyzed (% of area).

three feather tips were collected from three SL chickens without and seven with vitiligo, laid parallel in the same orientation in an aluminum cup containing O.C.T, and snap frozen with liquid nitrogen. Longitudinal sections (6 µm) were cut at −19°C, fixed in acetone and subjected to indirect immunohistochemical staining.

vitiligo state, “NV”, “EV”, “AV” or “CV” means feather tips were from SL chickens that never developed SLV (n = 3), from vitiliginous SL chickens (n = 7) just (<2 weeks) before, during or after SLV development.

⁴

data are shown as mean ± SE. a,b Data in the same row without common letters are different at P ≤ 0.05.

⁵

T cells include both alpha-beta and gamma-delta TCR+ T cells.