Bright field microscopy

Boyden Chamber assay was performed to access invasion capability of breast cancer cells according to earlier reported method with slight modifications.^{15 (link)} Breast cancer cells were treated with CoCl2 (100 µM)) and transfected with IL-6R siRNA. Other groups were treated with Dia (6 µM) or 5-Aza (5 µM). These treated cells were placed on the upper chamber of Boyden set up. In lower chamber, VEGF containing medium was added. After 24 h of incubation, upper chamber was taken and non-migrated cells were swapped with cotton plug. Upper chambers were then fixed with methanol and stained with haematoxylin and eosin followed by proper washing. Migrated cells were subjected to bright field microscopy at ×20 magnifications (Carl Zeiss).

HM@WY-Lip/UA were fabricated through the generation of water-in-oil droplets using microfluidics [56 (link)]. The water phase consisted of a 5 wt% mixture of HAMA, WY-Lip/UA, and a photoinitiator (in a ratio of 80:12:8, w/w/w). Additionally, a mixture consisting of paraffin oil and 5 wt% Span 80 was formulated for the oil phase. The oil and water phases were combined and introduced into the microfluidic device through the inlet utilizing a syringe pump, using a flow rate ratio that had been adjusted appropriately. Cross-linking was achieved by exposing the droplets to UV radiation for 5 min. After this, the cross-linked microspheres underwent a cleansing process involving acetone and deionized water, followed by a freeze-drying period of 48 h.
Bright-field microscopy (ZEISS, Germany) was used to examine the morphology and size of HM@WY-Lip/UA. To validate the effective binding of liposomes to HM, liposomes labeled with DiI were used to confirm the successful incorporation of liposomes, which were subsequently observed utilizing LSCM. After 60 s of gold spraying using an ion sputtering instrument, the surface morphology and microstructure of HM or HM@WY-Lip/UA were examined through SEM analysis (FEI Sirion 200, USA). SEM images were obtained at an accelerating voltage of 10 kV.

Mammosphere formation assay was performed as described previously [5 (link)]. In brief, either 2500 cells/well or 1000 cells/well was grown in 24-well low-attachment plates. After 7 days, all mammospheres in each well were assessed via bright-field microscopy (Zeiss). Mammospheres were imaged and analyzed with ImageJ software to obtain the diameters. The mammospheres which diameter was larger than 50 μm were counted. Three independent experiments were performed.

C2C12 myotubes were treated with differentiation medium supplemented with 10% conditioned medium from KPC for 48 h. Pictures of myotubes were taken with bright field microscopy (Zeiss), and diameters of myotubes were measured using the software JMicroVision as previously described [16 (link)].

Paraffin human post mortem eye tissues were stained for iron by the Perls method. Briefly, Perls reaction staining was performed after deparaffinization by incubating slides in a solution containing 4% potassium ferrocyanide (Sigma) in 4% aqueous hydrochloric acid (Sigma) for 1 h at room temperature to yield a Prussian blue reaction product. Sensitivity for iron detection was enhanced by incubation in 3,3′-Diaminobenzidine Tetrahydrochloride (DAB, Vector Labs, Eurobio, Courtabouef, France) for 1 h at room temperature, producing a brown reaction product. Nucleus counterstaining was performed by incubation in a solution of 1% nuclear red (Merck) for 30 s. Stained sections were mounted in aqueous medium. Control of amplification was realized by incubation of sections with DAB without the preceding Perls reaction step. Sections were analyzed by brightfield microscopy (Zeiss, Rueil Malmaison, France) and pictures were acquired using identical exposure conditions in the optic nerve or at a similar distance from the optic nerve.