In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

The overall goal of this procedure is to observe the ultra structure of megakaryocytes located within the mouse bone marrow, and to quantify the different maturation stages using high-resolution transmission electron microscopy. The main advantage is the direct examination of megakaryocytes in the native environment, which differs from in vitro cultured megakaryocytes that as we know do not reach the complete level of maturation. After harvesting the tibia and femurs from 12 to 18-week-old C57BL/6 mice according to standard protocols, use a sharp razor blade to remove the epiphyses from each bone.

Holding each bone with tweezers, insert a 21 gauge needle attached to a five milliliter syringe filled with cacodylate buffer into one end of the bone and flush the bone marrow into a 15 milliliter collection tube containing two milliliters of fresh cacodylate buffer. Immediately after flushing, use a plastic pipette to transfer the bone marrow cylinders into one milliliter of fresh glutaraldehyde fixative solution for a 60-minute incubation at room temperature. For embedding of the bone marrow in agarose, wash the fixed samples in fresh cacodylate buffer and use a plastic pipette to carefully transfer the bone marrow to a glass slide.

Using a warm pipette, quickly apply a drop of 2%liquid agar to the bone marrow cylinders, and immediately place the slide on ice for one to two minutes. When the agar has solidified, use a stereo microscope and a sharp razor blade to discard the extremities of each bone marrow cylinder and transfer the trimmed marrow blocks to a 1.5 milliliter microcentrifuge tube containing one milliliter of cacodylate buffer. For resin embedding, fix the blocks with 1%osmium tetroxide in cacodylate buffer in a chemical hood for one hour at four degrees Celsius before washing the blocks one time with cacodylate buffer and one time with distilled water.

After the water wash, stain the blocks with 4%uranyl acetate in distilled water for one hour followed by two washes in distilled water as demonstrated. After the last wash, dehydrate the blocks through an ascending series of ethanol immersions and distilled water as indicated. To obtain a uniform infiltration and polymerization of the epoxy resin inside the marrow, incubate the blocks in two successive baths of propylene oxide for 15 minutes before incubating the samples in a one-to-one mixture of 100%propylene oxide and epoxy resin for one hour on a slow rotary shaker at room temperature.

At the end of the incubation, add 100%epoxy resin to the marrow blocks for an overnight incubation under the same conditions, then add 100%epoxy resin for another incubation of two hours. At the end of the incubation, use a microscope to orient the marrow blocks in flat silicone molds to permit their subsequent transverse sectioning and fill the molds with epoxy resin before placing them at 60 degrees Celsius for 48 hours. For ultra thin sectioning, mount the sample block onto an ultra microtome support and mount the support onto the sample holder.

Use a diamond milling cutter to trim the samples at a 45 degree angle to remove the excess resin around the tissue before using a diamond knife blade equipped with a water tank to cut transverse 500 and 100 nanometer thick sections for histological and TEM analysis respectively, then use a loop to transfer the 500 nanometer thick sections floating on the water surface onto a glass slide and to deposit the 100 nanometer thick sections onto 200 mesh thin bar copper grids with a paper filter underneath. For toluidine blue staining, after drawing the 500 nanometer thick sections on a 60 degree hot plate, add filtered 1%toluidine blue in distilled water to the sections for a one to two-minute incubation. At the end of the incubation, wash the samples with distilled water.

When the slides have dried, use mounting medium to mount the samples with a coverslip and view the samples by light microscopy. For contrast staining, label the 100 nanometer sections with 4%uranyl acetate for five minutes followed by three five-minute washes with distilled water. After the last wash, stain the sections with lead citrate for three minutes, followed by three five-minute washes in distilled water as demonstrated.

After the last wash, place the lower side of each grid in contact with a piece of filter paper first to allow the grids to be placed onto the filter paper to dry. To examine the sections by TEM, when the grids have dried, select a low magnification to assess the general quality of the preparations. To determine the number of megakaryocytes from each stage of maturation in each transverse section, select a higher magnification and quantify the number of stage I, II, or III megakaryocytes only in squares that are fully covered by tissue.

In this representative histological analysis, the compactness micro-vessel continuity and size and shape of the megakaryocytes can be clearly observed. Murine megakaryocytes are divided into four stages of maturation. Stage I megakaryocytes have a large nucleus.

The presence of the earliest detectable stage of the demarcation membrane system is also a key criterion of this stage. In stage II, granule formation begins and the development of the demarcation membrane system is initiated. Stage III megakaryocytes are giant cells with well-developed demarcation membrane systems, clearly defined cytoplasmic territories and peripheral zones devoid of organelles, and eccentrically located nuclei.

The pyrenocyte stage of megakaryocyte maturation is characterized by a naked nucleus. As illustrated, megakaryocytes are frequently observed in contact with endothelial cells. On occasion, the megakaryocytes form short invasive protrusions that penetrate the endothelium or they extend proplatelets into the sinusoidal lumen.

TEM also permits the visualization of ultra structural details, as well as the presence of hematopoietic cells that have been engulfed by the megakaryocytes. Bone marrow flushing has to be performed carefully and immediately after bone dissection. To maximize tissue integrity, the marrow is surrounded with a gel of agar before dehydration.

Following this procedure, the bone marrow blocks can be imaged at different magnifications or can be used for other 3D electron microscopy analysis, such as focused ion beam scanning electron microscopy.