Tissue sectioning infiltration

Fig. 4. (A and B) Breast tissue from the same patient, posttreatment. Section of the tumor bed posttreatment shows reactive changes consisting of cellular fibrous tissue with numerous vessels (granulation tissue) and infiltration by lymphocytes, plasma cells and numerous iron-loaded macrophages. No residual carcinoma cells are seen. (A) H E, magnification x200 (B) H E, magnification x400.

Figure 9 Treatment of operable brain tumors with BCNU-loaded, p(CPP SA)-based wafers. (A) Schematic cross sections through a brain The tumor is illustrated as a black circle (he surrounding tissue is infiltrated by cancer cells. Upon tumor resection, up to eight drug-loaded wafers (cylindrical disks) are placed into the resection cavity to minimize the risk of local tumor recurrence. (B) Overall survival of 222 patients with recurrent brain tumors (phase HI clinical trial, after adjustment for prognostic factors). (C) Overall survival of 240 patients with newly diagnosed brain tumors (phase HI clinical trial, including results from (he long-term follow-up). Abbreviation ITT, intent-to-treat Source From Refs. 58 and 63 (B and C).

Alkaline phosphatase labeling not only is useful as a second double stain but also may be preferred for tissues rich in endogenous peroxidase, such as bone marrow or lymphoid tissue containing infiltrating myeloid cells, particularly when using frozen sections. Because complete blocking of endogenous peroxidase in blood... 

Cerroni L, Smolle J, Soyer HP, et al. Immunophenotyping of cutaneous lymphoid infiltrates in frozen and paraffin-embedded tissue sections a comparative study. / Am Acad Dermatol. 1990 22 405-413. 

Frozen fixed tissue is sectioned in a cryostat also known as a microtome in a freezer. The tissue is fixed in 4% paraformaldehyde, rinsed, and then cryoprotected by infiltration in 20% sucrose in buffer with agitation overnight at 4°C (cold room). After 24 h the tissue blocks will sink in the solution, indicating that they are infiltrated. This infiltration step is critical. If skipped, the tissue will freeze with damaged cells and holes from ice crystals, and the resulting tissue sections on microscope slides will be brittle and might crack. The tissue must be fixed first to hold the cells in place, as cryoprotection and freezing without fixation will destroy the tissue. 

Freezing microtome - used for cutting 15- to 50-p.m thick sections of fixed tissue after infiltrated with 20% sucrose and quickly frozen with liquid CO2 sections are for floating incubations. 

Histological scoring of tissue sections uses the following criteria. 0 = no inflammation, 1 = focal inflammatory infiltration, and 2 = severe and difHise inflammatory infiltration (Fig. 2). Note It is recommended that two fields per slide, one around the instep area and the other around the Achilles tendon, be used to quantitate arthritis or neutrophil numbers. Severe and diffuse inflammatory infiltration is defined as inflammatory infiltration in the both the instep and Achilles tendon per slide. Focal inflammatory infiltration is defined as inflammatory infiltration in one field per slide.)... 

In preparation for paraflSn infiltration water and fats are removed after fixation from the small blocks of tissue by consecutive extractions with alcohol and fat solvents, such as xylene. The tissue blocks are then placed in several changes of molten paraffin. After the displacement of xylene by paraffin is complete, the tissue blocks in paraffin are removed from the oven and hardened by cooling. Sections can be cut easily by a rotary microtome at a thickness of 5 to 7 microns (0.005 to 0.007mm.). When water-soluble carbohydrates are to be studied, it is important to cut and mount the paraffin sections on slides without exposure to water. In ordinary work, the paraffin ribbons are floated on water and lifted on slides for mounting. Paraffin is removed from the tissue sections prior to microchemical tests by consecutive baths in several changes of xylene and alcohols. 

Fig.1 Schematic view of flat-infiltration and flat-embedding procedure for array tomography. The tissue is fiat-infiitrated on top of an inverted glass petri dish containing a few drops of LR White, then covered by a glass coverslip (upper panel), and infiltrated overnight at 4 °C. On the next day, the tissue is flat embedded between a glass slide and two sheets of ACLAR plasBc (lower panel). One of the plastic sheets is a spacer while the other Is a cover. The tissue section is deposited in the hole of the perforated spacer sheet that was previously glued to one side of the glass slide (/owerpane/). Then, a few drops of LR White are added to the tissue section, and then covered by the cover sheet (lower panel). The tissue is incubated at 55 X for 24 h for polymerization...

Each specimen was dehydrated, infiltrated and embedded in Technovit based methylmethacrylate. One section was cut and around in preparation for scanning electron microscopy (SEM). In each case, three overview photos were necessary and four high magnification fields (40X) were photographed and digitized. These fields were later analyzed for volume fraction of soft tissue, bone... 

Brorson SH, Andersen T, Haug S, Kristiansen I, Risstubben A, Tchou H, Ulstein J (1999) Antigen retrieval on epoxy sections based on tissue infiltration with a moderately increased amount of acceleratorto detect immune complex deposits in glomerular tissue. Histol Histopath 14 151 155... 

Participation of the T-cell subsets was also examined histochemically in subcutaneously rechallenge tumor sections, Fig. (6)-Protocol C. The accumulation of lymphocytes in the tumor tissues and around the tumor increased in the CVS-treated group, 2 days after the s.c. tumor rechallenging, Fig. (8)-B. Immunohistochemical staining revealed increased infiltration of mature CD3 (D), CD4 (F), and CD8 (H) -positive T-cell s into the tissue in the CVS group. The tumor mass disappeared in about 50 percent of the CVS-treated mice 7 days after tumor inoculation, while in all control PBS mice it increased progressively until death (data not shown). 

Fig. (8). Increased infiltration of CD3, CD4 and CDS positive cells into the s.c. rechallenge tumor following CVS treatment [44]. BALB/c mice were injected s.c. with 5 x lCr Meth A cells in the right and left flanks on day 0 and 9, respectively. CVS (50 mg/kg) (B,D,F,H) or PBS (A,C,E,G)was injected i.t. into the primary tumor 3 times every 2 days from day 2. Recnallenge tumor sections, including subcutaneous tissues, were obtained 2 days after tumor rechallenge and stained with hematoxylin-eosin (A,B) or immunohistochemically (C-H). a Subcutaneous muscle layer, b interstitial space, c tumor tissue.

Small pieces of tissue are fixed for 1 hr in a mixture of 4% formaldehyde and 0.5% glutaraldehyde in 100 mM phosphate buffer (pH 7.4) (Chicoine and Webster, 1998). They are infiltrated with 2.3 M sucrose for 24 hr at 4°C, mounted with specimen pins (Leica, Deerfield, IL), and frozen by immersion in liquid nitrogen. The frozen tissue is sectioned at -110°C using an Ultracut E ultramicrotome (Leica) equipped with a diamond knife and an FC4 cryoattachment. Cryosections -60-80 nm thick are thawed and then mounted on Formvar-carbon-coated grids. 

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