protocol for
Immunostaining using Indirect Immunofluorescence
in Dr. Barres' lab
1) Prepare "antibody" buffer:
Distilled water 200 ml
NaCl (150 mM) 2.25 g
Tris Base (50 mM) 1.5 g
BSA (Sigma A2153) (1%) 2.0 g
L-Lysine (100 mM) 3.6 g
Azide (0.04%) Add 2 ml of a 4% stock in water
Adjust to pH 7.4 (pH paper) About 6 ml 1 M. HCl
Store at 4o C indefinitely.
2) Transfer cover slips to staining box. Gently transfer coverslips from 24-well plate to staining box (a homemade box with 24 flat topped pedestals glued to its bottom to support coverslips and keep them raised above the botom which will have some water for humidification; it should also have a top to prevent evaporation and keep slides humidified) using a coarse pair of forceps (keep cell side face up!). All volumes below are 50-100 ul unless otherwise specified, but you can use as little as 25 ul if necessary.
3) Fixation. 4% paraformaldehyde for 10 minutes (unless for BrdU staining or other fix-sensitive antigens, then use 60-90 seconds maximum). (For intracellular antigens such as GFAP can use instead a 10 minute fix in -20o C acid-alcohol (5% glacial acetic acid plus 95% ethanol) or in 100% methanol.; whenever alcohol fixation is used the triton permeabilization step can be skipped.) Paraformaldehyde cross links proteins so that they do not solubilze when the cells are permeabilized (below). By contrast, alcohol fixation works not by cross linking but by precipitating the proteins.
After the 10 minute fix step, pick up the coverslip with forceps and rinse it in three washes of PBS.
4) Block Non-Specific Binding. Incubate in 50% goat serum diluted in the "antibody" buffer for 30 minutes. The goat serum contains high levels of goat antibodies and proteins which bind non-covalently to non-specific binding sites and also bind covalently to reactive aldehyde groups created by the paraformaldehyde. Without this step, the primary antibody would bind both to these reactive aldehyde groups and the non-specific binding sites and create high levels of background staining. (Goat serum is used because the fluorochrome-conjugated secondary antibodies (see below) are raised in goats; this minimizes any unwanted binding of the secondary antibody to the goat antibodies that bind to the non-specific binding sites.)
After the block step, drain the coverslip, but it is not necessary to rinse it in PBS.
5) Permeabilization. Triton X-100 (0.4%; which is 1:50 of a 20% stock in water) for 30 minutes. This step can be combined with step #4 by adding the triton to the goat serum solution. Do not permeabilize when surface staining is being performed, particularly for galactocerebroside (a glycolipid) staining or A2B5 (a ganglioside) staining. These lipid antigens would be solublized by triton.
After permeabilization, rinse the coverslip three times in PBS (particularly for alcohol fixes).
6) Primary antibody incubation. Incubate the coverslip in the primary antibody solution for 30 minutes (or 60 minutes to overnight if necessary). To prepare this solution, dilute the primary antibody with the "antibody" buffer. The best dilution depends on the concentration of primary antibody and the density of the antigen. Generally supernatants are used neat or diluted up to 1:10. Ascites are used at 1:100 to 1:1000, as are polyclonal antisera. (The optimal dilution can be determined in a separate experiment.) Note: typical supernatant antibody concentration is 1-10 ug/ml, ascites concentrations are 1-10 mg/ml, as are polyclonal antisera. Thus typical final primary concentrations are about 1-10 ug/ml. (When the best concentration is not known and a guess must be made, I generally guess to use the supernatant undiluted, the ascites at 1:100, and polyclonals at 1:500. But beware of the "prozone" effect especially with IgM monoclonal antibodies).作者: 园丁## 时间: 2012-3-13 12:41
Rinse the slip three times in PBS.
7) Secondary antibody incubation. Incubate the coverslip in the secondary antibody solution for 30 minutes (or 60 minutes to 2 hours if necessary). To prepare this solution, dilute the secondary antibody with the "antibody" buffer. The dilution depends on the lot and concentration of the secondary antibody, the density of the antigen, and to some extent the concentration of the primary antibody that was used. The proper dilution must generally be established in a separate experiment. Generally the secondary antibodies will be used at a final concentraton of about 1-10 ug/ml (our stock aliquots are about 1-2 mg/ml). Generally I use a dilution of about 1:100 for secondary antibodies. For single-label staining use FITC as a fluorochrome rather than rhodamine or Texas Red.
Rinse three times in PBS.
8) Optional: Postfix in acid-alcohol for 10 minutes--particularly helpful for high density cultures, to stabilize the antibody binding and to decrease the likelihood that the cells will detach during coverslipping. Can't hurt (unless using phycoerythrin coupled secondaries, which it destroys) and generally improves the appearance.
Rinse three times in PBS.
9) Mount in PBS-glycerol based mountant such as Citifluor (or VectaShield; Vector) by inverting the coverslip onto a drop of the mountant on a 25 x 75 mm glass microscope slide (labelled on frosting with pencil only). Gently blot excess with a Kimwipe. Paint clear nail polish around the edges. Allow to air dry for 10 minutes before examining under the microscope. Can store slides for several weeks in the dark in a 4o C refrigerator.
Citifluor is a mixture of PBS and glycerol and contains a proprietary substance that helps retard fluorescein bleaching, but isn't so helpful for rhodamine or Texas Red bleaching. VectaShield works for both. N-paraphenylenediamine (0.1%) is useful for FITC bleaching too (but is carcinogenic). N-propylgallate (0.4% or higher for confocal studies) retards rhodamine and Texas-Red bleaching, but doesn't help for FITC. Note that FITC has a steep pH optimum, being maximally bright between pH 8 and 9. Outside this range its intensity is many times lower.作者: 园丁## 时间: 2012-3-13 12:41
Immunostaining Troubleshooting
1) No staining:
-- fix sensitivity (used wrong fix),
-- antibodies are off (e.g. if contaminated by bugs),
-- used the wrong secondary antibody (e.g. used an anti-mouse antibody to detect a rabbit primary).
2) High background staining:
-- failed to block non-specific binding (either too little goat serum or too short an incubation)
-- secondary antibody concentration too high
-- primary antibody concentration too high
-- the secondary antibody may specifically detect antigens on the tissue stained
-- the primary or secondary antibody may be binding to Fc receptors expressed by some types, particularly macrophages or microglia
3) Specific staining is weak:
-- Primary antibody concentration is too low
-- Secondary antibody concentration is too low
-- Density of antigen is low. If using a monoclonal, consider using a polyclonal if possible, or move to an immunostaining technique with a higher amplification: biotin-conjugated secondary antibodies followed by streptavidin-FITC or an immunoenzymatic technique such as peroxidase methods (secondary antibody conjugated to HRP or use the ABC or PAP methods).作者: 园丁## 时间: 2012-3-13 12:42
Immunostaining Controls
1) Never do immunostaining without appropriate controls. Negative controls must be set up for every labelling experiment; often other controls are also required.
2) Negative controls You must establish that when the primary antibody is omitted or replaced with an irrevant primary antibody of the same type and concentration that there is no staining.
3) Positive controls. If there is no specific staining observed, show that the labelling protocol used would have worked where than antigen is already known to be expressed.
4) Preabsorbtion controls. Particularly when staining with antibodies to a peptide or other antigen available in purified form, show that preabsorbtion of the primary antibody with 100 (or more) molar excess of the purified antigen eliminates the staining.
5) Double Label controls. When doing double labelling, show that the secondary antibodies each recognize specifically only the appropriate primary antibody. Also show that the filter set used does not recognize Texas Red on the FITC channel and vice versa.作者: jkobn 时间: 2012-3-13 12:42
Re:在观察的时候,一般是把盖玻片(爬片)放在载玻片上,请问是将盖玻片(爬片)有细胞的一面朝上还是朝下?
Please see the answer from " Curren Protocol in Cell Biology": Label slides and place 1 drop of mounting medium onto slide. Pick up coverslip from well , gently blot off excess PBS by touching the edge to a Kimwipe, then invert coverslip , cell-side-down , onto drop. Gently blot mounted coverslip with paper towel, then seal edge of coverslip onto slide by painting the edge with a rim of nail polish. Let dry.
So should be cell-side-down, if you put it otherwise, then you are wasting your time.作者: guagua 时间: 2012-3-13 13:36
I have ever encountered the same problem as you. I think that is because something wrong with the fixative. Do you use freshly-made 4% paraformaldehyde? If not, I suggest you make fresh fixative each time.
As to the coverslip, I usually added antibodies to the clean parafilm then inverted the coverslip(cell surface down) to the parafilm. This way we can save antibodies. For a 2X2cm2 coverslip, 50ul antibodies will be enough.作者: yhz1973 时间: 2012-3-13 13:40
The coverslip( cell surface down) cover and touch the antibody solution. Do you have " Current Protocols in Cell Biology"? The method was described in detail in the book.
As to the first question, I'd like to hear other people's opinion.
Sorry about the typing, because something wrong with the chinese system in my computer, I have to use English.作者: fqswdzd 时间: 2012-3-13 13:41
One thing left, I put the coverslip in the multi-well cell culture dish( according to the coverslip size ), all the wash was in the dish.
I think if your coverslip is coated properly, the cells shouldn't get off easily.作者: tangxin_80 时间: 2012-3-13 13:41
For immunofluorescence staining, the negative controls usually have some kinds of fluorescence, but much weaker than the positive staining. As to the reasons, I think, the most important one is tissue autofluorescence. In my hands, the fibrous tissues usually have stronger autofluorescence. And that is one of the reasons that we must do negative control along with the samples.作者: cocacola 时间: 2012-3-13 13:47
一般细胞免疫荧光，有条件的很多人已经用流式做了。本人头一次接触细胞免疫荧光的实验，有很多问题要问。
细胞的爬片我不是很懂，看来是让细胞在处理好的盖玻片上存活，我现在有multi well micro slide，高压消毒，用polylysine处理后，是否可以让细胞爬片？有人做过吗？悬浮和贴壁的细胞都可以使用吗？如何加入处理因素？很着急，请大家指点！谢谢！
主任，我查到的文献上免疫组化染色步骤是这样的，
Cryostat sections were prepared and fixed in acetone,and endogenous peroxidase was blocked byincubation with a 1:100 dilution of H2O2 in methanol.Sections were washed in phosphate-buffered saline(PBS) and then incubated with the primary antipleiotrophinantibody (which was used in the ELISA[goat anti-human pleiotrophin]) diluted to 5 mg/mL(or a 1:20 dilution) in PBS containing 1% bovineserum albumin at room temperature (for 2 hours) orat 4 °C (overnight). After subsequent washing (three 2-minute washes) in PBS, the secondary antibody was added (peroxidase-coupled rabbit anti-goat IgG[Dianova, Hamburg, Germany]) in 10% type ABO human serum and incubated for 1 hour at room temperature.After a further wash in PBS, peroxidaseactivity was localized by staining with diaminobenzidine as a substrate (Vector Laboratories, Burlingame, CA). The sample then was rinsed in water (two 2-minute washes), and nuclei were stained with hematoxylin (30–60 seconds). After a final rinse in water, the samples were dried and covered.
有几个问题要请教一下，
1.该文献上加一抗前，并未加动物血清封闭，可以吗？
2.该文献上一抗是用血清稀释液稀释的吗？
3.二抗peroxidase-coupled rabbit anti-goat IgG 是腊根过氧化物酶标记的吗？其中coupled 是什么意思？
4.secondary antibody in 10% type ABO human serum该如何做？作者: 伊莎贝拉 时间: 2012-3-13 14:33
主任，我查到的文献上免疫组化染色步骤是这样的，
Cryostat sections were prepared and fixed in acetone,and endogenous peroxidase was blocked byincubation with a 1:100 dilution of H2O2 in methanol.Sections were washed in phosphate-buffered saline(PBS) and then incubated with the primary antipleiotrophinantibody (which was used in the ELISA[goat anti-human pleiotrophin]) diluted to 5 mg/mL(or a 1:20 dilution) in PBS containing 1% bovineserum albumin at room temperature (for 2 hours) orat 4 °C (overnight). After subsequent washing (three 2-minute washes) in PBS, the secondary antibody was added (peroxidase-coupled rabbit anti-goat IgG[Dianova, Hamburg, Germany]) in 10% type ABO human serum and incubated for 1 hour at room temperature.After a further wash in PBS, peroxidaseactivity was localized by staining with diaminobenzidine as a substrate (Vector Laboratories, Burlingame, CA). The sample then was rinsed in water (two 2-minute washes), and nuclei were stained with hematoxylin (30–60 seconds). After a final rinse in water, the samples were dried and covered.
有几个问题要请教一下，
1.该文献上加一抗前，并未加动物血清封闭，可以吗？
2.该文献上一抗是用血清稀释液稀释的吗？
3.二抗peroxidase-coupled rabbit anti-goat IgG 是腊根过氧化物酶标记的吗？其中coupled 是什么意思？
4.secondary antibody in 10% type ABO human serum该如何做？
To the first question: In this kind of articles, the authors only briefly mentioned their methods. After all, immunostaining is basic technique. And that they didn't mention doesn't mean they didn't do.
To the second question: they mentioned clearly that the primary antibody is diluted in PBS containing 1% bovine serum albumin.
To the third question: Yes. couple:偶联.作者: glass 时间: 2012-3-13 14:36
Sometimes the lysosome was broken and enzymes came out of the lysosome. If this happened, you will find your staining not specifically distributed in the cytoplasm. Maybe you should try to optimize your sample preparation step.作者: jrwyyplt 时间: 2012-3-13 14:38
冷冻切片和培养细胞的免疫组化染色，需注意的方面是忌使用3%双氧水阻断内源性过氧化物酶，如果使用想一想双氧水伤口消毒的样子，那冷冻切片和培养细胞能使用3%双氧水吗？一样的效果。另外在切片和细胞干燥后应用丙酮和甲醇等量混合固定液固定，效果好，冷冻切片也不要太厚，6,7微米就可以了作者: one 时间: 2012-3-13 14:41
图片附件: 68527480.jpg (2012-3-13 13:09, 30.1 KB) / 该附件被下载次数 12
