Adipose-derived stem cell stimulation using poly-L-lactic acid injection

Table 1 Material information for histological preparation, antibody validation and final labeling

	Material information
Glycine	Fisher scientific	212485
Normal donkey serum	Innovative Research, Inc.	IGDNSER500ML-36672
Goat serum	Gibco	16210064
TBS (10 ×), pH = 7.4	Quality Biological	351-086-491
TritonX-100	Thermo Scientific	A16046.AP
Xylene substitute-3	StatLab	7400-1
100% reagent alcohol	StatLab	6900-1
95% reagent alcohol	StatLab	9500-1
70% reagent alcohol type 1 water blend	StatLab	7070-1
Citraconic anhydride	Thermo Scientific Chemicals	L05238.14
PCNA monoclonal antibody (10D10E11)	Thermo Fisher Scientific	60097-1-IG
CD90 monoclonal antibody (2D7D11)	Thermo Fisher Scientific	66766-1-IG
Endoglin/CD105 polyclonal antibody	Thermo Fisher Scientific	10862-1-AP
CD31 monoclonal antibody (2H8)	Invitrogen	MA3105
Adiponectin monoclonal antibody (OTI3G10), TrueMAB™	Thermo Fisher Scientific	TA804768
DLK1 polyclonal antibody	Thermo Fisher Scientific	29288-1-AP
Β-catenin monoclonal antibody (CAT-5H10)	Invitrogen	13-8400
PPARγ monoclonal antibody (1F4A2)	Thermo Fisher Scientific	66936-1-IG
Perilipin-1 (D1D8) XP® rabbit mAb #9349	Cell Signaling Technology	9349
Anti-CD68 antibody (EPR20545) - goat IgG (Chimeric)	Abcam	ab289671
Anti-CD80 antibody (RM1133)	Abcam	ab315832
CD163 recombinant rabbit monoclonal antibody (JA51-30)	Thermo Fisher Scientific	MA5-32684
DAPI	Invitrogen	D3571
Donkey αGoat 594	Invitrogen	A11058
Donkey αGoat 647	Invitrogen	A32849
Donkey αRabbit 647	Invitrogen	A31573
Goat αMouse IgG1 488	Invitrogen	A21121
Goat αMouse IgG1 647	Invitrogen	A21240
Goat αMouse IgG2a 594	Invitrogen	A21135
Goat αRabbit 555	Invitrogen	A21428
Goat αRabbit 594	Invitrogen	A11012
Goat αRabbit 647	Invitrogen	A21245
Fluoro-Gel (with Tris buffer)	Electron Microscopy Sciences	17985-10

TBS: Tris-buffered saline; PCNA: Proliferating cell nuclear antigen; DLK1: Delta-like non-canonical Notch ligand 1; PPAR-γ: Peroxisome proliferator-activated receptor gamma; IgG: Immunoglobulin G; DAPI: 4’,6-diamidino-2-phenylindole.

Table 2 Antibody information, concentration gradients, optimal concentration, and respective positive control tissues for antibody validation

Antibody	SKU#	Concentration gradient	Optimal concentration	Control tissue
PCNA	60097-1-IG	1:500, 1:1000, 1:2000	1:500	Human tonsil
CD90	66766-1-IG	1:250, 1:500, 1:1000	1:250	Human hippocampus
CD105	10862-1-AP	1:1000, 1:2000, 1:4000	1:1,000	Human testis
CD31	MA3105	3 μg/mL, 9 μg/mL, 15 μg/mL	15 μg/mL	Human lung
Adiponectin	TA804768	1:50, 1:100, 1:200	1:50	Human adipose tissue
Pref-1	29288-1-AP	1:100, 1:200, 1:500	1:100	Human placenta
β-catenin	13-8400	1:100, 1:500, 1:1000	1:100	Human tonsil
PLIN1	66936-1-IG	1:50, 1:100, 1:200	1:100	Human adipose tissue
PPARγ	9349	1:250, 1:500, 1:1000	1:250	Human tonsil
CD68	ab289671	1:500, 1:1000, 1:2000	1:500	Human lung
CD80	ab315832	1:50, 1:100, 1:200	1:100	Human lymph
CD163	MA5-32684	1:50, 1:100, 1:200	1:50	Human lung

PCNA: Proliferating cell nuclear antigen; Pref-1: Preadipocyte factor 1; PLIN1: Perilipin 1; PPAR-γ: Peroxisome proliferator-activated receptor gamma.

Table 3 Experimental labeling parameters for primary antibodies at the respective validated concentrations and secondary labels for primary study

Primary antibody	Vendor and SKU	Primary concentration	Secondary antibody	Vendor and SKU	Secondary concentration
Panel 1
PCNA	Thermofisher, 60097-1-IG	1:500	Goat αMouse IgG1 488	Invitrogen, A21121	1:500
CD105	Thermofisher, 10862-1-AP	1:1000	Goat αRabbit 555	Invitrogen, A21428	1:500
CD90	Thermofisher, 66766-1-IG	1:250	Goat αMouse IgG2a 594	Invitrogen, A21135	1:500
CD31	Invitrogen, MA3105	15 μg/mL	Donkey αGoat 647	Invitrogen, A21447	1:500
Panel 2
β-catenin	Invitrogen, 13-8400	1:100	Goat αMouse IgG1 488	Invitrogen, A21121	1:500
Adiponectin	Thermofisher, TA804768	1:50	Goat αMouse IgG2b 594	Invitrogen, A21145	1:500
Pref-1	Thermofisher, 29288-1-AP	1:100	Goat αRabbit 647	Invitrogen, A21245	1:500
Panel 3
PLIN1	Cell Signaling Technology, 9349	1:100	Goat αRabbit 594	Invitrogen, A11012	1:500
Panel 4
PPAR-γ	Thermofisher, 66936-1-IG	1:250	Goat αMouse IgG1 647	Invitrogen, A21240	1:500

PCNA: Proliferating cell nuclear antigen; Pref-1: Preadipocyte factor 1; PLIN1: Perilipin 1; PPAR-γ: Peroxisome proliferator-activated receptor gamma; IgG: Immunoglobulin G.

Table 4 Experimental labeling parameters for primary antibodies at the respective validated concentrations and secondary labels for macrophage analysis (group 2)

Primary antibody	Vendor and SKU	Primary concentration	Secondary antibody	Vendor and SKU	Secondary concentration
Panel 1
CD68	Abcam, ab289671	1:500	Donkey αGoat 594	Invitrogen, A11058	1:500
CD80	Thermofisher, 66406-1-IG	1:100	Donkey αRabbit 647	Invitrogen, A31573	1:500
Panel 2
CD163	Thermofisher, MA5-32684	1:50	Goat αRabbit 647	Invitrogen, A21245	1:500

Table 5 Exposure times for the imaged channels from the client tissue for each panel for group 1, all positive and secondary controls were exposed to same exposure for respective panels

Sample	Exposure times (millisecond)
	350 nm	488 nm	555 nm	594 nm	647 nm
Panel 1	0.75	30	400	375	50
Panel 11	0.75	30	125	175	35
Panel 2	1	20	N/A	75	175
Panel 3	1	N/A	N/A	25	N/A
Panel 4	0.75	N/A	N/A	N/A	120
Exposure times for the imaged channels from the submitted tissue for each panel for set 22
Panel 1	0.5	-	-	45	80
Panel 2	0.5	-	-	18	12

¹Indicates exposures for samples labeled in a second labeling round.

²All positive and secondary controls were exposed to same exposure for respective panels.

N/A: Not applicable.