Ix73 inverted microscope

Knocked down SH-SY5Y cells were transfected on day
5 (Table 1), using an equal, maximum recommended,
dose of ancestral or mutant LRSAM1 construct. The
empty vector (pIRES2-EGFP) was used as a control to
test for any possible effect of the construct on the cells.
Untransfected-B cells were also used as controls which
were treated with LRSAM1 siRNA but not transfected with
either ancestral or mutant LRSAM1 construct. Cells were
monitored using the IX73 inverted microscope (Olympus,
Japan) before and 48 hours after transfection.

IHC staining on formalin-fixed, paraffin-embedded tissue sections was performed as before (18 (link)). Briefly, slides were deparaffinized and rehydrated, epitope retrieval was performed in a Retriever 2100 (Aptum Biologics) with R-Buffer A (Electron Microscopy Sciences), and endogenous peroxidases/phosphatases were quenched with BLOXALL blocking solution (Vector Laboratories). Tissues were blocked with Animal-Free Blocker R.T.U. (Vector Laboratories), probed with anti-Luciferase antibodies (Abcam; AB185923, 1:200 dilution) overnight at 4°C, washed with PBS, and incubated with ImmPRESS anti-Rabbit polymer detection reagent (Vector Laboratories) for 30 minutes at room temperature. Visualization was performed by incubation with 3,3′-diaminobenzidine (DAB; Vector Laboratories), tissues were counterstained with Gill No.3 Hematoxylin (Sigma), coverslipped, and imaged on an Olympus IX73 inverted microscope.

293T cells were transduced with Adenoviral vectors in 8-well chamber slides (NEST Scientific 1162B43). After 24 hours, cells were fixed with 10% formalin, washed with PBS, blocked/permeabilized in Animal-Free Blocker containing 0.3% Triton X-100, and incubated overnight with Rabbit anti-Luciferase (Abcam; ab185923, 1:500 dilution) or Chicken anti-GFP antibodies (Aves Labs; GFP-1020, 1:500 dilution) at 4°C. The next day, wells were washed, secondary staining was performed with goat anti-rabbit AF594 (Invitrogen; A-11012, 1:1,000) or donkey anti-chicken AF488 (Jackson Immunoresearch; 703–545–155, 1:1,000) conjugated antibodies, counterstained with DAPI, mounted with VECTASHIELD, and imaged on an Olympus IX73 inverted microscope.

After clamping the renal pedicle at P22 the left kidney was quickly removed, rapidly weighed, sectioned coronally in two parts and placed in microcentrifuge tubes and frozen in liquid nitrogen. The right kidney was also removed after clamping of the renal pedicle, quickly weighed, and rapidly cut coronally. One-half of this right kidney was fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS, and the other half was placed in RNAlater (Qiagen, Waltham, MA) for future preparation of cDNA. A section of liver was also obtained for each mouse and placed immediately in liquid nitrogen.
After overnight fixation at 4°C, the kidney tissues were washed in PBS, quenched in NH₄Cl and further washed in PBS. The samples were then placed in 10% neutral buffered formalin (VWR, Radnor, PA, United States) at 4°C for 16–18 h. The fixed kidney samples were then dehydrated in serial alcohols, then cleared with xylene, embedded in paraffin, and cut into 4-μm sections on a rotary microtome at the Keck School of Medicine of USC Norris Pathology Core. The fixed tissues were stained with hematoxylin and eosin (H&E) for histological evaluation. We then obtained images using an Olympus IX73 inverted microscope using a Plan Achromat × 2 objective for a total magnification of × 20 (numerical aperture of 0.06 and working distance of 5.8 mm).