How Many Immune Fluorescence Pits Did You Win?

You Will Always See The Beautiful Pictures Of Cells Extracted By Others Waving In Front Of You. However, It Is Your Turn To Find That It Is Not As Simple As Preparing Cells - Fixing Cells - Cell Permeability - Sealing - Primary Antibody - Primary Antibody - Secondary Antibody - Nuclear Staining - Microscopic Examination. Later, It Was Found That The Experimental Price, Which Can Be Described In No More Than 50 Words, Is So Difficult& Nbsp; Five Suggestions Let You Avoid The Pit Of Immunofluorescence, And Meimei Should Show Her Own Achievements, Which Is Also Right Because She Lost A Lot Of Hair In The Experiment. 1. Consider The Recommended Dilution Of Antibody To Achieve A High Signal To Back Ratio. If The Antibody Concentration Is Too Low, The Fluorescence Signal Is Weak And May Be Difficult To Distinguish From The Background, But If The Antibody Concentration Is Too High, It Will Lead To Higher Background Staining. " 2. Washing With Physiological Solution Can Remove Unbound Fluorescent Reagents, Or Those Fluorescent Reagents That Are Only Stuck To The Target But Not Specifically Bound, So As To Improve The Signal To Back Ratio. 3. Fluorescent Materials Must Be Carefully Stored At The Recommended Temperature And Always Protected From Light To Protect Their Spectral Integrity. 4. When Designing Multiple Experiments, You Should Consider The Unique Properties Of Each Fluorescent Group, Such As The Maximum Absorption Wavelength And Maximum Emission Wavelength, Extinction Coefficient And Stokes Shift, And Other Factors To Make Sure That Your Reagents Match The Instruments Used; 5. There Must Be A Control. In The Primary Cell Immunofluorescence Test, It Is Generally Recommended To Use Cells Or Tissues That Do Not Express The Target As A Negative Control. In Fluorescent Western Blot Or ELISA, The Protein Or Peptide Containing The Target Antigen Is Suitable As A Positive Control. The Extracted Primary Cells Generally Use Immunofluorescence To Detect Several Major Markers Of Cells To Determine The Authenticity Of Cells, Such As Cell Systems' Primary Aortic Cells, CD 62E (E-Selectin), VWF/Factor VIII, Di-I-Ac-LDL Three Major Markers, As Well As Standard Tests For Ex Warehouse, No HIV, BPV And Mycoplasma Detection. Some Common Pitfalls: 1. The Signal Is Weak Or No Signal, And There Are Too Few Stained Cells. The Following Points Can Be Considered: 1. The Solution To The Problem That The Target Protein Is Not Expressed In Cells: Prepare Cell Lysate, And Use Western Blotting To Verify Whether The Target Protein Is Expressed In Cells. 2. The Solution To The Problem That There Are Too Few Cells Expressing The Target Protein: Use More Cells In The Sample, And Try Other Transient Transfection Methods, Or Use A Stable Transfection Cell Line 3. Poor Cell Permeability Increases The Time Or Concentration Of The Penetrant, Or Use Other Penetrants 4. The Fixation Steps Before Dyeing Destroy The Antigen Epitope And Use Other Fixation Methods 5. Antibody Problem The Antibody Is Not Suitable, The Wrong Antibody Selection Or The Non-standard Antibody Dilution Will Also Lead To No Signal, So It Is Recommended To Dilute The Same Sample According To The Standard, And Make The First Antibody Dilution Curve According To The Best Amount Of The Second Antibody, To Determine The Optimal Dilution Ratio Of Primary Antibody; 2、 There Are Scattered Light Spots Between Cells In The Field Of Vision: A: 1. Non Specificity Caused By Too Little PBS When Taking Photos; PBS Is Not Filtered, And The Undissolved Residue Adheres, Resulting In; 3、 Under The Microscope, Only The Blue Light Of DAPI Is Observed, And The Target Fluorescence Is Almost As Weak As That Of Abstainers? It Is Likely That The Antibody Proportion Is Too Low, And The Antibody Concentration Needs To Be Increased, Which Can Be Coordinated With The Increase Of The Secondary Antibody Concentration; Confocal Excitation Fluorescence Intensity Is Not Large Enough; Whether The Secondary Antibody Is The Corresponding Species When Incubated& Nbsp; 4、 There Are Always Some Blue Dots Around The Nucleus? This Situation Is Generally Caused By Mycoplasma Contamination. It Is Recommended That Mycoplasma Killing Drugs Be Used For Detection 2 Weeks Later. Those Who Abstain From Using Mycoplasma Can Cover Mycoplasma Fluorescence By Increasing The Background Value Of The Confocal Machine.