Table 3.

Lipofuscin Identification and Subtraction from total and colocalized FMRP puncta

				Total Puncta (#)			Total Colocalized Puncta (#)
Figure	Circuit ROI	Donor	Nociceptor Marker	647-Puncta	Auto in 647	FMRP	Colocalized Puncta	Auto in Marker	Colocalized FMRP
5	Dorsal Root	Male 2	Nav1.7	41,288	145	41,143	13,731	241	13,490
			Peripherin	41,288	145	41,143	11,421	75	11,346
		Female 1	Nav1.7	4,909	138	4,771	1,684	115	1,569
			Peripherin	4,909	138	4,771	1,230	95	1,135
6	Dorsal Root	Male 2	TRPV1	17,647	118	17,529	16,638	136	16,502
		Male 3		29,604	380	29,224	12,641	816	11,825
		Female 1	TRPV1	8,738	37	8,701	4,919	0	4,919
		Female 2		20,317	145	20,172	5,972	27	5,945
	Lateral Tracts	Male 2	TRPV1	76,835	5,506	71,329	52,916	2,357	50,559
		Male 3		9,374	430	8,944	4,978	136	4,842
		Female 1	TRPV1	2,632	302	2,330	565	0	565
		Female 2		28,265	1,143	27,122	9,676	208	9,468
	Laminae I-II	Male 2	TRPV1	229,810	11,967	217,843	150,729	15,179	135,550
		Male 3		597,268	55,619	541,649	395,242	46,309	348,933
		Female 1	TRPV1	40,119	4,763	35,356	15,528	381	15,147
		Female 2		384,771	17,335	367,436	143,254	2,651	140,603
7	Dorsal Root	Male 2	CGRP	829	237	592	459	391	68
		Male 3		738	95	643	226	226	0
		Male 4		8,070	41	8,029	1,998	51	1,947
		Female 1	CGRP	18,199	88	18,111	8,375	124	8,251
		Female 3		20,760	61	20,699	8,478	63	8,415
		Female 4		11,868	113	11,755	8,881	133	8,748
	Laminae I-II	Male 2	CGRP	46,911	9,021	37,890	23,192	9,864	13,328
		Male 3		35,838	7,995	27,843	12,907	10,461	2,446
		Male 4		99,891	36,108	63,783	37,811	25,138	12,673
		Female 1	CGRP	76,827	14,120	62,707	47,450	13,542	33,908
		Female 3		55,012	7,374	47,638	30,254	6,282	23,972
		Female 4		319,063	23,468	295,595	138,096	11,100	126,996

^*Note: Lipofuscin were detected in both the FMRP and nociceptor marker channels by quantifying colocalization with either 561- (Figures 6 and 7) or 405- (Figure 5) induced autofluorescence using the same algorithm parameters to detect FMRP puncta. This was used to estimate the number of puncta arising from lipofuscin in channels of interest per FOV. This number was then subtracted from the evident number of puncta present in each FOV to more accurately identify punctate FMRP immunoreactivity. The example puncta numbers given are the overall sums detected and identified per donor for each quantitative analysis.