Table 1. Immunocytochemistry Protocols.

antibody	fixative solution	postfix	block	primary	wash	secondary
Fibroblast growth factor receptor, FGFR (mouse)	M/F (9∶1) on ice; ON, −20°C	No	0.1% T, 0.1% A, 2% BSA in TBS; 1 hr, RT	2 ul in 500 ul in block; ON, RT	0.1% T in TBS	2 ul Cy3 Goat anti-mouse IgG+IgM in 500 ul block; ON, RT
HSPG (mouse)	M/F (9∶1) on ice; ON, −20°C	No	0.1% A, 2% BSA in PBS; 1 hr, RT	1 ul in 500 ul in block; ON, RT	0.1% T in PBS	2 ul Cy3 Goat anti-mouse IgG+IgM in 500 ul block; ON, RT
Activated FGFR, pFGFR (rabbit)	M/F (9∶1) on ice; ON, −20°C	No	0.1% T, 0.1% A, 2% BSA in TBS; 1 hr, RT	1 ul in 500 ul block; ON, RT	0.1% T in TBS	2 ul Cy3 Goat anti-rabbit in 500 ul block; ON, RT
Activated Epidermal growth factor receptor, pEGFR (rabbit)	2.5% P, 1% G, 1% SMB, in 0.1 M cacodylate buffer, pH 7.2; ON, 4°C after microwave	NaBH4	0.1% T, 0.1% A, 2% BSA in TBS; 1 hr, RT	1 ul in 500 ul block; ON, RT	0.1% T in TBS	2 ul Cy3 Goat anti-rabbit in 500 ul block; ON, RT
Manduca Fasciclin II, C3 (mouse)	4% P or 4%P, 0.1% G, in 0.1 M PB	No	0.5% T, 0.1% A, 2% BSA in TBS; 1 hr, RT	1∶10,000 in 500 ul block; ON, RT	0.1% T in TBS	2 ul Cy3 Goat anti-mouse IgG+IgM in 500 ul block; ON, RT
Phospho-histone H3 (rabbit)	4% P in 0.1 M PB; ON, 4°C	DNAse	0.5% T, 0.1% A, 2% NGS in PBS; 1 hr, RT	1.5 ul in 500 ul block; ON, RT	0.1% T in PBS	2.5 ul Cy3 Goat anti-rabbit F(ab)2 in 500 ul PBS with 0.1% A, 0.1% T; 3–4 hrs, RT
Ankyrin B (mouse)	2.5% P, 1% G, 1% SMB in 50 mM carbonate buffer (pH 9.4), final pH 10.8; ON, 4°C after microwave	NaBH4	0.1% T, 0.1% A, 2% BSA in TBS; 1 hr, RT	2 ul in 500 ul block; ON, RT	0.1% T in TBS	2 ul Cy3 Goat anti-mouse IgG+IgM in 500 ul block; ON, RT, then 2 ul Alexa 564-Donkey anti-goat in 500 ul block, ON, RT

Microwave protocol: 18°C; Power level 2; 2 min on/2 min off/2 min on/2 min off.

Pella research-grade oven (# 3450, with variable power controller #3430 and cold spot).

A: sodium azide.

Alexa 564-Donkey anti-goat tertiary (Molecular Probes #A11056).

BSA: bovine serum albumin, IgG-free (Jackson Immunoresearch #001-000-161).

Cy3-Goat anti-mouse secondary (Jackson #115-165-068).

Cy3-Goat anti-rabbit secondary (Jackson # 111-165-144).

Cy3-Goat anti-rabbit F(ab)₂ (Jackson # 111-166-047).

DNAse I (Sigma): 10 U DNAse, 4 mM MgCl2 in PBS; 1 hr, 37°C.

G: glutaraldehyde.

M/F: methanol/37% formalin.

NaBH₄: 0.01 M NaBH₄, 0.5% SMB in 0.05 M Tris HCl, pH 7.5; 30 min.

NGS: normal goal serum.

ON: overnight on a shaker.

P: paraformaldehyde.

PB: phospate buffer pH 7.4.

PBS: phosphate-buffered saline (10 mM sodium phosphate, 150 mM NaCl, pH 7.4).

RT: room temperature.

SMB: sodium metabisulfite.

T: Triton X-100.

TB: 10 mM Tris-HCl, pH 7.4.

TBS: Tris-buffered saline (20 mM Tris-HCl, 150 mM NaCl, pH 7.4).