TABLE 1.

Troubleshooting table.

Step	Problem	Possible reason(s)	Solution
Steps 1-34	Incomplete hardening of the resin tissue blocks	Incomplete tissue dehydration and/or resin infiltration	Check the temperature of reagents for each step and extend timing as needed
		Inadequate exposure of blocks to ultraviolet light or failure to keep the cryochamber temperature within correct range	Check that the ultraviolet light source of the chamber is intact and increase the frequency of temperature monitoring to maintain a range between -10 °C to -20 °C for 24-48 h
	Nonspecific gold label (i.e., over non-tissue regions such as the lumen of a vessel or capillary)	Blocking serum is inappropriate for the antibody used	If appropriate, adjust the blocking serum
		Primary antibody is inappropriate for the tissue	Check that the antibody is appropriate, and the expiry date and dilution of the antibody and gold solution used
	Gold label is low or absent	Antibody failed to bind antigenic sites	Run a dilution experiment to establish the optimal antibody concentration
		Gold solution failed to bind antibody-labeled sites	Complete a run on tissue that is a known positive control
	Background debris (dirt) is high	Contaminated solutions	Add additional rinsing steps and ensure that all solutions are filtered immediately before use
Steps 35-56	Cell sample does not become red-transparent	Incomplete lysis of RBCs	Centrifuge at 300g at 4 °C (with brake) for 5 min to remove the supernatant and repeat the lysing step for 1-2 min at RT
	Fluorescence signal is poor/inadequate	Instrument malfunction (i.e., a weak excitation signal is produced)	Use an alternate machine to determine if the problem is related to the hardware or reagents
		Antibody/reagents are inappropriate or used at wrong concentration	If an instrument is malfunctional, use another cytometer; if not, check and repeat the experimental and staining procedures
	Inaccurate/shifting compensation matrix indicated by variable readings for nonspecific IgG-staining tubes for blood or tissue	Compensation matrix settings have changed	Run single-color controls, or alternating-color controls, to reset and confirm that the compensation matrix is accurate

● TIMING

Steps 1-8: 48 h to dehydrate and infiltrate lung tissue blocks (36-48 per rodent group) with resin

Steps 9-13: 58 h to polymerize tissue blocks in Unicryl resin, label and archive them

Steps 14-16: 8 h to cut 3-4 tissue blocks, that is, 2 h to rough-cut 1 tissue block, and prepare 1-μm-thick sections on glass slides and (approximately 15-20) 90-nm-thick sections on Formvar-coated grids

Steps 17-20: 16 h to treat (approximately 15-20) 90-nm-thick sections with primary antibody

Steps 21-26: ~2-3 h to label 15-20 grids/sections with pA-AU; 3 h to treat approximately 15-20 grids/sections with silver halide (Box 1)

Steps 27-31: 4 h to enhance electron-density of cell membranes and organelles in 15-20 grids/sections

Steps 32-34: ~2-3 d to view multiple grids/sections by TEM, collect overviews and determine the appropriate analysis. Allow an additional ~3 h to collect 100 images for qualitative or quantitative analysis, and 2 min per cell to quantify antigenic sites using the macro program

Steps 35-40: ~2 h to prepare cell suspensions from blood

Steps 41-48: ~2 h to prepare cell suspensions from lung tissue

Steps 49-56: 5 h to immunostain cells and 30 min to analyze six tubes by flow cytometry after a compensation matrix has been established