COX staining solution:

50 mM phosphate (pH 7.4),

4 mM 3,3’-diaminobenzidine,

2 μg/ml catalase,

200 μM cytochrome c,

84 mM malonate,

60 μM rotenone,

and 4 mM antimycin A.

SDH staining solution:

50 mM phosphate (pH 7.4),

42 mM succinic acid,

0.4 mM phenazine methosulfate,

0.5 mM nitro blue tetrazolium,

4.5 mM EDTA,

60 μM rotenone,

4 mM antimycin A,

and 2 mM KCN.

COX inhibitor solution:

COX staining solution + 2 mM KCN

SDH inhibitor solution:

SDH staining solution + 84 mM malonate

Procedure (at room temperature):

COX/SDH dual staining: skip steps 3 to 5 for single COX staining

1. COX staining solution incubation for 30 min,

2. 3 x 5 min washes in 50 mM phosphate (pH 7.4),

3. SDH staining solution incubation for 10 min,

4. 2 x 5 min washes in 50 mM phosphate (pH 7.4),

5. 2 x 5 min washes in 50 mM phosphate (pH 7.4),

6. incubation in 80% glycerol in 50 mM phosphate (pH 7.4).

The negative controls using COX inhibitor solution and SDH inhibitor solution were performed with each batch of staining. Brightfield germarium images were collected by Zeiss Axio Observer Z1 microscope.