To determine relative chemokine mRNA levels, we used the Riboquant
Multiprobe RNase Protection Assay system (PharMingen, San Diego, CA).
Briefly, draining (cervical) lymph nodes and eyeballs containing
conjunctival samples were removed from the mice immediately after they
were killed. The conjunctiva was dissected from its attachments to the
eyelids. The tissues were placed in 2 ml of RNAStat 60, homogenized,
and immediately frozen on dry ice. The samples were stored at −80°C
until used. For use, separate RNA samples from five mice per
experimental group were defrosted on ice, and the RNA was extracted
with a phenol chloroform procedure using DEPC-treated materials.
Aliquots of the samples were run on a 1% agarose gel to determine
concentration and ascertain any potential degradation. The samples were
then used as per manufacturer’s instructions with the mCK-5 probe
positive for lymphotaxin (Ltn), Regulated on activation normal T cell
expressed (RANTES), eotaxin, macrophage inflammatory peptide-1α
(MIP-1α), MIP-1β, MIP-2, interleukin-gamma–induced protein 10
(IP-10), monocyte chemotactic protein-1 (MCP-1), T cell activation
gene-3 (TCA-3), and the housekeeping genes L32 and GAPDH. To
semiquantify differences in amounts of RNA, the film was scanned after
development, and the bands were subjected to densitometry using Digital
Science ID software (Kodak). The numerical values were normalized
according to the densitometry of the housekeeping genes GAPDH and L32.