Snap-frozen tissue

Methods are under development for analysis of protein from formalin-fixed tissues, although protein yield may be less than that from frozen tissue. Extraction of high-yield, quality protein from formalin-fixed tissue is generally difficult because of the extensive cross-links formed in formalin-fixed samples. Currently, the optimal tissue specimen for use with protein lysate microarrays is fresh tissue, immediately embedded in a cyroprotectant solution and frozen or snap-frozen tissue. 

As a gold standard, fresh tissue prepared by snap-frozen method, cut by cryostat, and fixed in acetone, ethanol, or other non-cross-linking fixatives, has been generally accepted as reliable. 

Compound (Miles Laboratories, Elkhart, IN), snap-frozen, and cut into sections for comparison with paraffin-embedded cell sections (3) FFPE Cell Blocks Six cell pellets were fixed in 10% neutral buffered formalin immediately after harvest, at room temperature for 6,12,24h, 3,7, and 30 days, respectively. For further comparison with the cell model system, recently collected sample of human breast cancer tissues were processed by OCT-embedding and snap-freezing the corresponding routine FFPE block that was obtained from the Norris Cancer Hospital and Research Institute at the University of Southern California Keck School of Medicine (USC). This tissue block was processed routinely (formalin-fixed 24h and processed by automatic equipment). 

The sample materials from which proteins for proteomics studies may be extracted include fresh or snap-frozen cells from varied sources such as biological fluids, (serum, urine, plasma) and solid tissues such as biopsy specimens. Moreover, proteins isolated from ethanol-fixed paraffin-embedded tissues can be utilized for MS analysis.2 Protocols for the identification of proteins from formalin-fixed paraffin-embedded (FFPE) tissues have been recently developed.3 4 FFPE materials are the most common forms of biopsy archives utilized worldwide, and represent an important advancement for the large-scale interrogation of proteins in archival patient-derived materials. Finally, laser capture microdissected tissues have been successfully used for MS analysis.45... 

In most cases, fixation may be carried out at room temperature. Duration of formalin fixation depends on the nature and the size of the specimen, and may vary from 15 min to 24 h. Longer fixation may be associated with a partial loss of the antigenicity of the component of interest. After formalin fixation, tissue samples are washed in three changes of the buffered saline (PBS) from 15 min to 2 h, but not longer than 24 hours on the whole, since the formaldehyde fixation is partially reversible. After washing in PBS, specimens may be either snap-frozen in liquid nitrogen for subsequent cryosectioning, or dehydrated and embedded in paraffin or synthetic resin. 

For cryosectioning, tissue samples are quickly frozen with or without freezeembedding medium (e.g., Tissue Tek Miles Laboratories) and stored at 80°C until analysed. Optionally, aldehyde prefixation can also be used for tissue and organ probes before snap-freezing. Cutting of frozen tissue blocks is performed with a cryostat (a microtome mounted in a freezing cabinet). 

KCl solution (1.15 %, V=5 v/w of tissue) snap-frozen fresh liver tissue (25 g) ice bath... 

If the tissues have not been appropriately snap frozen, ice crystals will be present in the tissue, identifiable as slits or fracture lines through the tissue. Artifactual staining may occur along such fracture lines. Therefore, ice crystal damage should be avoided. The tissues should always be snap frozen. If freezing is performed slowly over dry ice, ice crystals will accumulate in the tissue. 

Tissue specimens are snap-frozen in liquid nitrogen for 30 sec immediately after removal and then transferred to a cryostat (Kammerer et al., 2001). Serial frozen sections of 5 pan thickness are cut and placed on silane-coated slides. They are air-dried for 30 sec, fixed in acetone for 1 min at room temperature (22°C), and air-dried at 22°C (Fig. 6.11). The sections are incubated with primary antibody in the antibody diluent (Dako) for 3 min by placing the slide horizontally on a hot plate at 37°C. (All incubation steps are carried out by placing the slide horizontally on the hot plate at 37°C.) Following a brief rinse in TBS, the sections are incubated with the goat-anti-mouse EnVision-HRP-enzyme conjugate for 3min at 37°C. 

Tissue specimens are snap-frozen at -60°C in an isopentane freezing bath (Neslab, Portsmouth, NH) (Resnick et al., 1995). Sections (5 pan thick) are cut on a cryostat,... 

An alternative to homogenization is sonication. In this technique, the tissue sample is snap frozen and then immediately ground to a fine powder using a mortar and pestle... 

Depending on the research question, other organs such as spleen and lymph nodes can be removed from the mice for (immunological) analysis, fixed in 4% formalin, and subsequently in 70% ethanol before paraffin embedding. Alternatively, the tissue, placed in aluminum cryotubes, can be snap frozen immediately in liquid nitrogen after dissection. This procedure should be performed if RNA, DNA, or protein needs to be isolated from the tissue. 

Most PNS antibodies can be detected using immunohistochemistry performed on cryostat sections of rat nerve tissue, usually snap frozen in iso-penthane [52, 155, 156] or fixed in formaldehyde for detection of CRMP-5 antibodies [52]. If the patient serum produces a staining pattern in nervous tissue consistent with an onconeural antibody at a dilution of 1 500 or more, this is usually regarded as positive [157] (Fig. 1). The specificity of... 

The following tissues are removed in the order listed below with a fresh set of clean instruments for each organ. The tissues (at least 0.5 g sample for each tissue-weight not required for protocol fulfillment) are snap frozen in liquid nitrogen and stored at or below -70 °C ... 

If the tissues have not been appropriately snap frozen, ice crystals will be present in the tissue, identifiable as slits or fracture lines through the... 

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