David Chan May 2, 2015

Histology Group Project: Report

For our group project, we selected the kidney. The first two images are paraffin-

embedded toluidine blue sections taken with the microtome.

In this first image, we are located within the cortex of the kidney, indicated by the

presence of the renal corpuscle which contains the Bowman’s capsule and the glomerulus. The

circular structure on the top of the image is the glomerulus surrounded by the Bowman’s capsule.

On the bottom left we believe is a blood vessel, most likely a vein due to the thin layer of tunica

media as opposed to the thick tunica media of the artery. Looking closely, you can see the

flattened nuclei surrounding the lumen of the vein which indicates the presence of the tunica

intima composed of simple squamous epithelia. The rest of the image is composed of tubules

which make up the kidney. The dark blue stain in the middle of the image is a tear within the

paraffin section.

This second paraffin-embedded toluidine blue section is a close up of several glomeruli,

the most notable being in the lower portion of the image. In this image you can see the density

of nuclei within the glomerulus indicated by the darkly stained dots. These nuclei are most

likely those of the podocytes. This image also shows the Bowman’s capsule and the darkly

stained flattened nuclei which surround the Bowman’s space. These nuclei are most likely those

of the simple squamous epithelium making up the parietal layer of the renal corpuscle. There

were issues with our toluidine blue slides with the darkness and clarity of the stains. Due to this

issue we weren’t able to see more distinguishable features around the renal corpuscle such as the

proximal and distal convoluted tubules.

Next we’ll examine our immunohistochemistry images which were stained for actin,

type-4 collagen and nuclei. We used Phalloidin to stain for f-actin in green, mouse-Texas red-

conjugated goat anti-mouse IgG to stain for type-4 collagen in red, and DAPI to stain for nuclei

in blue. With these markers we predicted we would see actin staining of the podocytes,

glomerulus, smooth muscle, skeletal muscle, and microvilli of the proximal convoluted tubules

in green. We also predicted for type-4 collagen to stain the basal lamina of the structures in the

kidney in red and for DAPI to stain the nuclei of cells in blue.

In this first image we see a blood vessel next to what we believe to be a glomerulus. The

blood vessel is most likely that of an artery due to the thick band of actin surrounding the lumen

of the vessel. The actin composite to the right shows a prominent thick green band surrounding

the lumen. This band is most likely that of the tunica media which is composed of smooth

muscle that would be stained green with our actin marker. There is also staining of the

glomerulus in green which is indicative of actin within the podocytes. In our collagen composite

image you can see the basal lamina surrounding the major structures in the image, particularly

those of the two glomeruli and the artery. In our DAPI composite there is a density of nuclei

within the glomerulus, most likely marking the nuclei of the podocytes. There is also staining in

the surrounding tunica media of the vessel which makes us believe that they’re the nuclei of the

smooth muscle. You can also see the nuclei within the innermost part of the vessel overlapped

by the collagen and actin staining, possibly the nuclei of simple squamous epithelium. In our

DAPI composite you can see the nuclei are arranged in a thick circular orientation which makes

us believe that the vessel is indeed an artery and not a vein. Although the blood vessel is

collapsed, it could be due to the sectioning process which could have flattened it.

This is our second composite image. Both of these images are of the same structure

taken at different focal planes. We believe this image is of a blood vessel, again we believe it is

an artery due to the large thick green band of actin around the lumen of the vessel. Arteries have

a thick layer of tunica media composed of smooth muscle while veins have a thin layer. You can

also see the basal lamina in the composite indicated by the bright red staining of type-4 collagen.

Upon examination of the DAPI composite, you can see the thick amount of nuclei surrounding

the lumen which we believe to be the nuclei of the smooth muscle of the tunica media. In the

actin and collagen composites you can see a wavy pattern which we believe to be the inner

elastic membrane of the artery. The bottom left structure of the image we believe to be a

glomerulus, although we aren’t certain due to the clarity of the image and the fact that the image

is cut off.

This is last image is also our best, and it’s that of the renal corpuscle. In this image you

can clearly see the branching of the glomerulus in the actin composite with the bright green

pattern of staining. In the type-4 collagen composite you can clearly see the surrounding basal

lamina of the glomerulus and the Bowman’s capsule. There is also a high density of nuclei

within the glomerulus where the podocytes are located.