Fix with 4 Paraformaldehyde

A second fixation with 4% paraformaldehyde in PBS is needed for 30 min. This step will insure that the labeled Fab remains bound to the correct U antibody. 

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Figure 7. A series of monoclonal antibodies raised against nuclear proteins. HeLa cells fixed with 4% paraformaldehyde were immunostained with monoclonal antibodies raised against nuclear proteins. The intracellular localization of these antigens are (a) dot inside the nucleolus, (b) whole nucleolus, (c) nuclear foci, (d) nucleoplasm, (e) the edge of the nucleus, (f) cytoplasm, (g) cytoskeleton, (h) plasma membrane, (i) mitochondria, (j) nucleus and cytoplasm, (k) nucleus and the paranuclear structure, and (1) paranuclear structure and nucleoplasm...

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

Tissue specimens are fixed with 4% paraformaldehyde and embedded in paraffin at 60°C for 1 hr. Sections (4 pm thick) are mounted on gelatin-coated glass slides, deparaffinized, and rehydrated in distilled water. They are treated with 0.005% pepsin for 15 min at 37°C, followed by heating in 0.01 M citrate buffer (pH 6.0) in a microwave oven (300 W) at 80°C for 15 min. The sections are washed in distilled water for 5 min, rinsed in 0.01 M PBS (pH 7.2) for 15 min, and treated with 0.3-1% H202 to quench endogenous peroxidase activity. 

The cells are fixed with 4% paraformaldehyde in PBS. Flow cytometry is performed on BD FACS Calibur Flow Cytometer. An example of results obtained from this analysis has recently been published (8). 

Optional) Plate an additional sample of the cell suspension at a cell density of 30,000 cells per glass coverslip and culture for 16 hr. Wash the cells with three changes of warm D-PBS and fix with 4% paraformaldehyde for 10 min at room temperature. 

Fig. 5. Subcellular localization of BARS. (A) To examine the localization of the individual CtBPs, COS7 cells were transfected with BARS (CtBP3/BARS), CtBPl, or CtBP2, and 24 h after transfection they were fixed with 4% paraformaldehyde for 10 min, and double stained with 1 g/ml affinity purified SNl antibody (green) and an anti-tubulin antibody (red). (B) (C) COS7 cells were transfected with BARS, and 24 h after the transfection they were fixed with 4% paraformaldehyde (B), or first permeabilized with SLO, incubated at 37° for 5 min, and fixed with 4% paraformaldehyde (C), for 10 min at room temperature. The cells were double-labeled with 1 iig/ml affinity-purified SNl antibody (red) and a monoclonal anti-giantin antibody (green) the merged images are also shown. The Golgi and plasma membrane localization of BARS are seen after SLO-permeabiUzation, which removes the soluble cytosolic pool of BARS.

Fix the inserts with 4% paraformaldehyde, and then stain with hematoxylin and eosin (H8cE) for 2 min or Diff-Quick staining solutions, according to the manufacturer s instructions. 

PFA fixative. Add 4% paraformaldehyde powder to PBS followed by 2-3 drops of 5 M NaOH. Dissolve PFA completely with vigorous shaking at 60°C. Allow to cool on ice before use. Make fresh as required see Note 2). 

Tissue specimens are fixed with 4% formaldehyde (freshly prepared from paraformaldehyde) for 12-18 hr at 4°C, and embedded in Epon (Groos et al., 2001). 

At P17, the pups are euthanized and their eyes enucleated and fixed for embedding and sectioning. Representative pups from each group are anesthetized and perfused through the left ventricle with 4% paraformaldehyde in 0.1 M sodium phosphate (pH 7.4) containing FITC-dextran for visualizing the retinal vasculature (see later). 

Acrolein - C3H4O (Fig. 3.4) (Sabatini et al., 1963) is an exceedingly fast penetrating chemical fixative that contains both an aldehyde and a double bond. Acrolein is used as a 2.5% solution along with 4% paraformaldehyde as a fixative solution. Warning - acrolein is highly poisonous, causes severe irritation to exposed skin, is extremely flammable, and is a mild carcinogen. Acrolein is NOT recommended because of its health and safety issues. 

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. 

B) Fix tissue - Perfuse the rat with 4% paraformaldehyde in phosphate buffer. After the animal is stiff to the touch, remove the kidney and place in fixative for dissection. Use a new sharp scalpel blade to cut the kidney, which is then submerged in the fixative solution for an additional 2 h. 

Wash cells in cold PBS and fix them with 4% paraformaldehyde (10 min, RT). 

In some cases, sequential perfusion with different fixatives has been used to aid preservation of peptide immunoreactivity. Typical examples include sequential perfusion with 4% paraformaldehyde in slightly acidic buffer, pH 6.5, followed by 4% paraformaldehyde in basic buffer, pH 9.0-11.0, or sequential perfusion with carbodiimide followed by paraformaldehyde alone or a carbodiimide/paraformaldehyde mixture (38),... 

Figure 2. Telencephalon of an adult newt and projections from olfactory and vomeronasal epithelia. (a) Horizontal sections of telencephalon. The head region was fixed in Bouin s fluid, cut at 10 pm and stained with Azan. NC, Nasal cavity. ON, olfactory nerve. TEL, telencephalon. Bar is 250 pm. (b) Projections from the main olfactory epithelium to the olfactory bulb corresponding to the boxed region (b) in Fig. 2a. Dil crystals were placed on the main olfactory epithelium for 5 days. The brain tissue was fixed in 4% paraformaldehyde, cut at 70pm and photographed under a fluorescent microscope. Bar is 250 pm. (c) Projections from the vomeronasal epithelium to the accessory olfactory bulb corresponding to the boxed region c in Fig. 2a. Dil crystals were placed on the vomeronasal epithelium for 5 days. Bar is 250 pm. The brain was treated as described above.

Animal tissue can be fixed in situ by perfusion with 4% paraformaldehyde, followed by immersion fixation of the dissected tissue (1-4 h, depending on the tissue size and fixation obtained with perfusion). This method is strongly recommended if brain or spinal cord tissues are used, as these tissues do not fix well by immersion only, owing to the poor penetration of the fixative in the tissue matrix. 

Tissues were fixed in 4% paraformaldehyde for 15-18 hr at 4°C or Ihr at room temperature. After being washed with TEST (50 mM Tris-HCl, 150 mM NaCl, 0.3%... 

Cells were plated onto coverslips and allowed to attach for 24 hours. After treated with 100 pM EGCG for 6 hours, ceUs were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X in PBS for 5 min CeUs were then washed with PBS, blocked with 2% bovine serum albumin for 30 min and incubated with p-FAK antibody for 1 hour at room temperature. After washing three times with 0.05% Triton X in PBS, cells were subsequently incubated with Alexa fluor 488 goat anti-rabbit second antibody for 1 hour at room teirqierature, and nuclei were stained with Topro 3 before mounting. The slides were viewed using a Zeiss LSM 510 laser confocal microscope (Carl Zeiss, Jena, Germai r). 

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